Antitumor therapy using ovine or bovine interferon-tau

ABSTRACT

The invention provides antitumor therapeutic methods employing bovine or ovine interferon-tau (IFN-τ) proteins and polypeptides. The IFN-τ proteins exhibit the antiviral and antiproliferative properties characteristic of type I interferons. An advantage of the invention is that IFN-τ has essentially no cytotoxic effects on treated cells as does, for example, IFN-α.

This invention was made with government support under NationalInstitutes of Health grants HD 10436, HD 26006, CA 38587, and CA 57084.Accordingly, the United States government has certain rights in thisinvention.

This application is a continuation of patent application Ser. No.08/438,753, filed May 10, 1995, now U.S. Pat. No. 5,705,363 hereinincorporated by reference, which is a continuation-in-part of patentapplication Ser. No. 08/139,891, filed Oct. 19, 1993, now abandoned,incorporated herein by reference, which is a continuation-in-part ofpatent application Ser. No. 07/847,741, filed Mar. 9, 1992, nowabandoned which is a continuation-in-part of application Ser. No.07/318,050, filed Mar. 2, 1989, now abandoned. Application Ser. No.08/139,891 is also a continuation-in-part of patent application Ser. No.07/969,890, filed Oct. 30, 1992 and now abandoned.

FIELD OF THE INVENTION

The present invention relates to interferon-τ compositions and methodsof use.

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BACKGROUND OF THE INVENTION

Conceptus membranes, or trophectoderm, of various mammals producebiochemical signals that allow for the establishment and maintenance ofpregnancy (Bazer, et al., 1983). One such protein, ovine trophoblastprotein-one (oTP-1), was identified as a low molecular weight proteinsecreted by sheep conceptuses between days 10 and 21 of pregnancy(Wilson, et al., 1979; Bazer, et al., 1986). The protein oTP-1 was shownto inhibit uterine secretion of prostaglandin F₂ -alpha, which causesthe corpus luteum on the ovary to undergo physiological andendocrinological demise in nonpregnant sheep (Bazer, et al., 1986).Accordingly, oTP-1 has antiluteolytic biological activity. The primaryrole of oTP-1 was assumed to be associated with the establishment ofpregnancy.

oTP-1 was subsequently found to (i) exhibit limited homology (50-70%)with interferon alphas (IFNα) of various species (Imakawa, et al.,1987), and (ii) bind to a Type I interferon receptor (Stewart, et al.,1987). Despite some similarities with IFNα, oTP-1 has several featuresthat distinguish it from IFNα including the following: oTP-1's role inreproductive biochemistry (other interferons are not known to have anyrole in the biochemical regulation of reproductive cycles), oTP-1'scellular source--trophoblast cells (IFNα is derived from lymphocytecells), oTP-1's size--172 amino acids (IFNα is typically about 166 aminoacids), and oTP-1 is weakly inducible by viruses (IFNα is highlyinducible by viruses). The International Interferon Society recognizesoTP-1 as belonging to an entirely new class of interferons which havebeen named interferon-tau (IFNτ). The Greek letter τ stands fortrophoblast.

The interferons have been classified into two distinct groups: type Iinterferons, including IFNα, IFNβ, and IFNω (also known as IFNαII); andtype II interferons, represented by IFNγ (reviewed by DeMaeyer, et al.).In humans, it is estimated that there are at least 17 IFNα non-allelicgenes, at least about 2 or 3 IFNβ non-allelic genes, and a single IFNγgene.

IFNα's have been shown to inhibit various types of cellularproliferation. IFNα's are especially useful against hematologicmalignancies such as hairy-cell leukemia (Quesada, et al., 1984).Further, these proteins have also shown activity against multiplemyeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi'ssarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinarybladder tumors and ovarian cancers (Bonnem, et al., 1984; Oldham, 1985).The role of interferons and interferon receptors in the pathogenesis ofcertain autoimmune and inflammatory diseases has also been investigated(Benoit, et al., 1993).

IFNα's are also useful against various types of viral infections(Finter, et al., 1991). Alpha interferons have shown activity againsthuman papillomavirus infection, Hepatitis B, and Hepatitis C infections(Finter, et al., 1991; Kashima, et al., 1988; Dusheiko, et al., 1986;Davis, et al., 1989).

Significantly, however, the usefulness of IFNα's has been limited bytheir toxicity: use of interferons in the treatment of cancer and viraldisease has resulted in serious side effects, such as fever, chills,anorexia, weight loss, and fatigue (Pontzer, et al., 1991; Oldham,1985). These side effects often require (i) the interferon dosage to bereduced to levels that limit the effectiveness of treatment, or (ii) theremoval of the patient from treatment. Such toxicity has reduced theusefulness of these potent antiviral and antiproliferative proteins inthe treatment of debilitating human and animal diseases.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to compositions of andmethods employing ovine interferon-τ. The invention includes an isolatednucleic acid molecule that encodes an ovine interferon-τ. One embodimentof this nucleic acid molecule is a nucleic acid molecule having thesequence presented as SEQ ID NO:1. In another embodiment, the nucleicacid molecule encodes an ovine interferon-τ polypeptide having asequence presented as SEQ ID NO:2. The ovine interferon-τ polypeptidemay include an amino-terminal extension, such as, a leader sequence.

In another embodiment, the present invention includes an expressionvector having a nucleic acid containing an open reading frame (ORF) thatencodes an ovine interferon-τ, including the nucleic acid andpolypeptide sequences described above. The vector further includesregulatory sequences effective to express the open reading frame in ahost cell. Further, the invention includes a method of recombinantlyproducing ovine interferon-τ using the expression vectors of the presentinvention. The expression vectors are introduced into suitable hostcells. The host cells are then cultured under conditions that result inthe expression of the ORF sequence.

In one embodiment, the present invention includes a recombinantlyproduced ovine interferon-τ protein.

Further, the invention includes a method of inhibiting tumor cellgrowth. In the method, the tumor cells are contacted with ovineinterferon-τ at a concentration effective to inhibit growth of the tumorcells. Target tumor cells include, but are not limited to carcinomacells, hematopoietic cancer cells, leukemia cells, lymphoma cells andmelanoma cells.

The invention also includes a method of inhibiting viral replication. Inthis method, cells infected with a virus are contacted with ovineinterferon-τ at a concentration effective to inhibit viral replicationwithin said cells. Ovine interferon-τ may be used to inhibit thereplication of both RNA and DNA viruses. Exemplary RNA viruses includefeline leukemia virus, ovine progressive pneumonia virus, ovinelentivirus, equine infectious anemia virus, bovine immunodeficiencyvirus, visna-maedi virus, and caprine arthritis encephalitis virus.

In a second aspect, the present invention relates to compositions of andmethods employing human interferon-τ's. In one embodiment, the inventionincludes an isolated nucleic acid molecule that encodes a humaninterferon-τ. Several variants of human interferon-τ (HuIFNτ) aredisclosed herein, including HuIFNτ1, HuIFNτ2, HuIFNτ3, HuIFNτ4, HuIFNτ5,HuIFNτ6 and HuIFNτ7. The nucleic acid molecules of the present inventioninclude nucleic acid molecules having the following sequences: SEQ IDNO:43, SEQ ID NO:29, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:27, SEQ IDNO:21 and SEQ ID NO:23.

The nucleic acids of the present invention also include nucleic acidmolecules encoding the following polypeptide sequences: SEQ ID NO:44,SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:22,and SEQ ID NO:24. The nucleic acids may further include sequencesencoding leader sequences for the human interferon-τ which they encode,for example, SEQ ID NO:41 or SEQ ID NO:42.

The second aspect of the invention further includes an expression vectorhaving a nucleic acid sequence containing an open reading frame thatencodes a human interferon-τ, including the nucleic acid and polypeptidesequences described above. The vector further includes regulatorysequences effective to express said open reading frame in a host cell.The regulatory sequence may include sequences useful for targeting orsecretion of the human IFNτ polypeptide: such sequences may beendogenous (such as the normally occurring human IFNτ leader sequences,present, for example, in SEQ ID NO:41) or heterologous (such as asecretory signal recognized in yeast, mammalian cell, insect cell,tissue culture or bacterial expression systems). In the expressionvector, regulatory sequences may also include, 5' to said nucleic acidsequence, a promoter region and an ATG start codon in-frame with thehuman interferon-τ coding sequence, and 3' to said coding sequence, atranslation termination signal followed by a transcription terminationsignal.

In a further embodiment, the invention includes a method ofrecombinantly producing human interferon-τ. In the method, theexpression vector, containing sequences encoding a human interferon-τopen reading frame (ORF), is introduced into suitable host cells, wherethe vector is designed to express the ORF in the host cells. Thetransformed host cells are then cultured under conditions that result inthe expression of the ORF sequence. Numerous vectors and theircorresponding hosts are useful in the practice of this method of theinvention, including, lambda gt11 phage vector and E. coli cells. Otherhost cells include, yeast, mammalian cell, insect cell, tissue culture,plant cell culture, transgenic plants or bacterial expression systems.

In another embodiment, the invention includes an isolated humaninterferon-τ protein or polypeptide. The protein may be recombinantlyproduced. Further, the protein or polypeptide may include any of thefollowing human interferon-τ sequences: SEQ ID NO:44, SEQ ID NO:30, SEQID NO:34, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:22, and SEQ ID NO:24.

The invention further includes a method of inhibiting tumor cell growth.In the method, the tumor cells are contacted with a human interferon-τpolypeptide at a concentration effective to inhibit growth of the tumorcells. The human interferon-τ may be a part of any acceptablepharmacological formulation. Tumor cells whose growth may be inhibitedby human interferon-τ include, but are not limited to, human carcinomacells, hematopoietic cancer cells, human leukemia cells, human lymphomacells, and human melanoma cells. In one embodiment, the tumor cells aresteroid-sensitive tumor cells, for example, mammary tumor cells.

In yet another embodiment of the present invention, human interferon-τpolypeptides are used in a method of inhibiting viral replication. Inthis method, cells infected with a virus are contacted with humaninterferon-τ at a concentration effective to inhibit viral replicationwithin said cells. The human interferon-τ may be a part of anyacceptable pharmacological formulation. The replication of both RNA andDNA viruses may be inhibited by human interferon-τ polypeptides.Exemplary RNA viruses include human immunodeficiency virus (HIV) orhepatitis c virus (HCV). An exemplary DNA virus is hepatitis B virus(HBV).

In yet another aspect, the present invention includes a method ofenhancing fertility in a female mammal. In this method, an effectivemammalian fertility enhancing amount of human interferon-τ isadministered to the female mammal in a pharmaceutically acceptablecarrier.

The invention also includes isolated human interferon-τ polypeptides.These polypeptides are derived from the interferon-τ amino acid sequenceand are typically between about 15 and 172 amino acids in length.

Also included in the invention is a fusion polypeptide that contains ahuman interferon-τ polypeptide that is between 15 and 172 amino acidslong and derived from a human interferon-τ amino acid coding sequence,and a second soluble polypeptide. In one embodiment, human interferon-τsequences are used in fusion constructs with other types of interferonsto reduce the toxicity of the other types of interferons, for example,interferon-α and interferon-β.

The invention also includes a polypeptide composition having (a) a humaninterferon-τ polypeptide, where said polypeptide is (i) derived from aninterferon-τ amino acid coding sequence, and (ii) between 15 and 172amino acids long, and (b) a second soluble polypeptide. Interferon-α andinterferon-β are examples of such second soluble polypeptides. Thiscomposition may be used to reduce the toxicity of the other types ofinterferons when the interferons are used in pharmaceutical formulationsor in therapeutic applications.

The invention also includes purified antibodies that are immunoreactivewith human interferon-τ. The antibodies may be polyclonal or monoclonal.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B present the nucleic acid coding sequence of a syntheticgene of OvIFNτ designed to include 19 unique restriction enzyme sitesspaced evenly throughout the coding sequence.

FIG. 2 shows the cloning strategy used for making a synthetic geneencoding OvIFNτ.

FIG. 3 shows a comparison of the predicted protein sequences of a humaninterferon-τ gene and an ovine interferon-τ gene. Divergent amino acidsare indicated by presentation of the alternative amino acid on the linebelow the nucleic acid sequences.

FIG. 4 presents data demonstrating that both OvIFNτ and IFNα were ableto drastically reduce growth of HL-60 cells.

FIG. 5 presents data demonstrating that rHuIFNα is cytotoxic and OvIFNτis not. In the figure, results of one of three replicate experiments arepresented as mean % viability±SD.

FIG. 6 presents the sequences of polypeptides derived from the IFNτsequence.

FIG. 7 presents the complete nucleic acid and amino acid sequence of anOvIFNτ sequence.

FIG. 8 presents data supporting the lack of cytotoxicity, relative toIFNα, when IFNτ is used to treat peripheral blood mononuclear cells.

FIG. 9 shows the results of treatment of a human cutaneous T celllymphoma line, HUT 78, with IFNτ.

FIG. 10 shows the results of treatment of a human T cell lymphoma line,H9, with IFNτ.

FIG. 11A presents data for the peptide inhibition, relative to FIV(feline immunodeficiency virus) replication, of polypeptides derivedfrom OvIFNτ with whole OvIFNτ. FIG. 11B presents data for the peptideinhibition, relative to HIV (human immunodeficiency virus) replication,of polypeptides derived from OvIFNτ with whole OvIFNτ.

FIG. 12 presents data demonstrating the inhibition of the antiviralactivity of IFNτ by IFNτ-derived peptides.

FIG. 13 presents data demonstrating the inhibition by IFNτ-derivedpeptides of OvIFNτ antiviral activity.

FIG. 14 presents data demonstrating the inhibition by IFNτ-derivedpeptides of bovine IFNα antiviral activity.

FIG. 15 presents data demonstrating the inhibition by IFNτ-derivedpeptides of human IFNα antiviral activity.

FIG. 16 presents data evaluating the lack of inhibition by IFNτ-derivedpeptides of bovine IFNγ antiviral activity.

FIG. 17 presents data demonstrating the anti-IFNτ-derived peptideantisera inhibition of the antiviral activity of IFNτ.

FIG. 18 presents data demonstrating the anti-IFNτ-derived peptideantisera inhibition of the binding of radiolabeled IFNτ to cells.

FIGS. 19A and 19B present an alignment of nucleic acid sequencesencoding IFNτ polypeptides.

FIGS. 20A and 20B present an alignment of amino acid sequences of IFNτpolypeptides.

FIG. 21 presents data comparing the cytotoxicity of IFNτ with IFNβ.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of a synthetic gene encodingovine interferon-τ (OvIFNτ). Also shown is the encoded amino acidsequence.

SEQ ID NO:2 is an amino acid sequence of a mature OvIFNτ protein.

SEQ ID NO:3 is a synthetic nucleotide sequence encoding a mature humaninterferon-τ (HuIFNτ) protein.

SEQ ID NO:4 is an amino acid sequence for a mature HuIFNτ1 protein.

SEQ ID NO:5 is the amino acid sequence of fragment 1-37 of SEQ ID NO:2.

SEQ ID NO:6 is the amino acid sequence of fragment 34-64 of SEQ ID NO:2.

SEQ ID NO:7 is the amino acid sequence of fragment 62-92 of SEQ ID NO:2.

SEQ ID No:8 is the amino acid sequence of fragment 90-122 of SEQ IDNO:2.

SEQ ID NO:9 is the amino acid sequence of fragment 119-150 of SEQ IDNO:2.

SEQ ID NO:10 is the amino acid sequence of fragment 139-172 of SEQ IDNO:2.

SEQ ID NO:11 is the nucleotide sequence of a natural HuIFNτ1 gene with aleader sequence.

SEQ ID NO:12 is the predicted amino acid coding sequence of the SEQ IDNO:11.

SEQ ID NO:13 is a 25-mer synthetic oligonucleotide according to thesubject invention.

SEQ ID NO:14 is a 25-mer synthetic oligonucleotide according the subjectinvention.

SEQ ID NO:15 is the amino acid sequence of fragment 1-37 of SEQ ID NO:4.

SEQ ID NO:16 is the amino acid sequence of fragment 34-64 of SEQ IDNO:4.

SEQ ID NO:17 is the amino acid sequence of fragment 62-92 of SEQ IDNO:4.

SEQ ID NO:18 is the amino acid sequence of fragment 90-122 of SEQ IDNO:4.

SEQ ID NO:19 is the amino acid sequence of fragment 119-150 of SEQ IDNO:4.

SEQ ID NO:20 is the amino acid sequence of fragment 139-172 of SEQ IDNO:4.

SEQ ID NO:21 is the nucleotide sequence of cDNA HuIFNτ6.

SEQ ID NO:22 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:21.

SEQ ID NO:23 is the nucleotide sequence of cDNA HuIFNτ7.

SEQ ID NO:24 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:23.

SEQ ID NO:25 is the nucleotide sequence of cDNA HuIFNτ4.

SEQ ID NO:26 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:25.

SEQ ID NO:27 is the nucleotide sequence of cDNA HuIFNτ5.

SEQ ID NO:28 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:27.

SEQ ID NO:29 is the nucleotide sequence of genomic DNA clone HuIFNτ2.

SEQ ID NO:30 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:29.

SEQ ID NO:31 is the nucleotide sequence, including leader sequence, ofgenomic DNA clone HuIFNτ3, a natural HuIFNτ gene.

SEQ ID NO:32 is the predicted amino acid sequence (including leadersequence) encoded by the sequence represented as SEQ ID NO:31.

SEQ ID NO:33 is the nucleotide sequence, excluding leader sequence, ofgenomic DNA clone HuIFNτ3, a natural HuIFNτ gene.

SEQ ID NO:34 is the predicted amino acid sequence of a mature human IFNτprotein encoded by HuIFNτ3, encoded by the sequence represented as SEQID NO:33.

SEQ ID NO:35 is the amino acid sequence of fragment 1-37 of SEQ IDNO:33.

SEQ ID NO:36 is the amino acid sequence of fragment 34-64 of SEQ IDNO:33.

SEQ ID NO:37 is the amino acid sequence of fragment 62-92 of SEQ IDNO:33.

SEQ ID NO:38 is the amino acid sequence of fragment 90-122 of SEQ IDNO:33.

SEQ ID NO:39 is the amino acid sequence of fragment 119-150 of SEQ IDNO:33.

SEQ ID NO:40 is the amino acid sequence of fragment 139-172 of SEQ IDNO:33.

SEQ ID NO:41 is the amino acid sequence of fragment 1-23 of SEQ IDNO:32.

SEQ ID NO:42 is the amino acid sequence of fragment 1-23 of SEQ IDNO:11.

SEQ ID NO:43 is the nucleotide sequence, excluding leader sequence, ofDNA clone HuIFNτ1.

SEQ ID NO:44 is the predicted amino acid sequence of a mature human IFNτprotein encoded by HuIFNτ1, encoded by the sequence represented as SEQID NO:43.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Interferon-τ (IFNτ) refers to any one of a family of interferon proteinshaving greater than 70%, or preferably greater than about 80%, or morepreferably greater than about 90% amino acid homology to either thesequence presented as (a) SEQ ID NO:2 or (b) SEQ ID NO:34. Amino acidhomology can be determined using, for example, the LALIGN program withdefault parameters. This program is found in the FASTA version 1.7 suiteof sequence comparison programs (Pearson, et al., 1988; Pearson, 1990;program available from William R. Pearson, Department of BiologicalChemistry, Box 440, Jordan Hall, Charlottesville, Va. ). Typically, IFNτhas at least one characteristic from the following group ofcharacteristics: (a) expressed during embryonic/fetal stages bytrophectoderm/placenta, (b) anti-luteolytic properties, (c) anti-viralproperties, and (d) anti-cellular proliferation properties. IFNτ can beobtained from a number of sources including cows, sheep, ox, and humans.

An interferon-τ polypeptide is a polypeptide having between about 15 and172 amino acids derived from an interferon-τ amino acid coding sequence,where said 15 to 172 amino acids are contiguous in native interferon-τ.Such 15-172 amino acid regions can also be assembled into polypeptideswhere two or more such interferon-τ regions are joined that are normallydiscontinuous in the native protein.

II. Isolation & Characterization of Interferon-τ

A. Ovine and Bovine Interferon-τ

1. Interferon-τ Coding Sequences

Ovine interferon-τ (OvIFNτ) is a major conceptus secretory proteinproduced by the embryonic trophectoderm during the critical period ofmaternal recognition in sheep. One isolate of mature OvIFNτ is 172 aminoacids in length (SEQ ID NO:2). The cDNA coding sequence contains anadditional 23 amino acids at the amino-terminal end of the matureprotein (Imakawa, et al., 1987). The coding sequence of this OvIFNτisolate is presented as FIG. 7.

Relative to other interferons, oIFNτ shares about 45 to 68% amino acidhomology with Interferon-α and the greatest sequence similarity with theinterferon-ωs (IFNωs) of about 68%.

For the isolation of OvIFNτ protein, conceptuses were collected frompregnant sheep and cultured in vitro in a modified Minimum EssentialMedium as described previously (Godkin, et al., 1982). Conceptuses werecollected on various days of pregnancy with the first day of matingbeing described as Day 0. OvIFNτ was purified from conceptus culturemedium essentially as described by Vallet, et al., (1987) and Godkin, etal. (1982).

The homogeneity of OvIFNτ was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Maniatis, et al.; Ausubel,et al.). Determination of protein concentration in purified OvIFNτsamples was performed using the bicinchoninic (BCA) assay (PierceChemical Co., Rockford, Ill.; Smith, et al., 1985).

A homologous protein to OvIFNτ was isolated from cows (BoIFNτ; Helmer,et al., 1987; Imakawa, et al., 1989). OvIFNτ and BoIFNτ (i) have similarfunctions in maternal recognition of pregnancy, and (ii) share a highdegree of amino acid and nucleotide sequence homology between matureproteins. The nucleic acid sequence homology between OvIFNτ and BoIFNτis 76.3% for the 5' non-coding region, 89.7% for the coding region, and91.9% for the 3' non-coding region. The amino acid sequence homology is80.4%.

Example 1 describes the reproductive functions of OvIFNτ. OvIFNτ andrecombinant human Interferon-α2 (rHuIFNα) were infused into uterinelumen of ewes at a variety of concentrations. The life span of thecorpus luteum was assessed by examination of interestrous intervals,maintenance of progesterone secretion, and inhibition of prostaglandinsecretion (Davis, et al., 1992). Comparison of the data resulting fromthese examinations demonstrated a considerable lengthening of theinterestrous interval when OvIFNτ is administered at 100 μg/day and nomeaningful effect when rHuIFNα is administered. These data support theconclusion that OvIFNτ significantly influences the biochemical eventsof the estrous cycle.

The antiviral properties of interferon-τ at various stages of thereproductive cycle were also examined (Example 2). Conceptus cultureswere established using conceptus obtained from sheep at days 12 through16 of the estrus cycle. Antiviral activity of supernatant from eachconceptus culture was assessed. Culture supernatants had increasingantiviral activity associated with advancing development of theconceptus up to Day 16 post estrus.

2. Recombinant Production of IFNτ

Recombinant OvIFNτ was produced using bacterial and yeast cells. Theamino acid coding sequence for OvIFNτ was used to generate acorresponding DNA coding sequence with codon usage optimized forexpression in E. coli (Example 3). The DNA coding sequence wassynthetically constructed by sequential addition of oligonucleotides.Cloned oligonucleotides were fused into a single polynucleotide usingthe restriction digestions and ligations outlined in FIG. 2. Thepolynucleotide coding sequence had the sequence presented as SEQ IDNO:1.

For expression of recombinant OvIFNτ, this synthetic coding sequence canbe placed in a number of bacterial expression vectors: for example,lambda gt11 (Promega, Madison, Wis.); pGEX (Smith, et al.); pGEMEX(Promega); and pBS (Stratagene, La Jolla, Calif.) vectors. Otherbacterial expression vectors containing suitable promoters, such as theT7 RNA polymerase promoter or the tac promoter, may also be used.Cloning of the OvIFNτ synthetic polynucleotide into a modified pIN IIIomp-A expression vector is described in Example 3. Production of theOvIFNτ protein was induced by the addition of IPTG. Soluble recombinantIFNτ was liberated from the cells by sonication or osmoticfractionation.

The protein can be further purified by standard methods, including sizefractionation (column chromatography or preoperative gelelectrophoresis) or affinity chromatography (using, for example,anti-OvIFNτ antibodies (solid support available from Pharmacia,Piscataway, N.J.). Protein preparations can also be concentrated by, forexample, filtration (Amicon, Danvers, Mass.).

The synthetic OvIFNτ gene was also cloned into the yeast cloning vectorpBS24Ub (Example 4; Sabin, et al.; Ecker, et al.). Synthetic linkerswere constructed to permit in-frame fusion of the OvIFNτ codingsequences with the ubiquitin coding sequences in the vector. Theresulting junction allowed in vivo cleavage of the ubiquitin sequencesfrom the OvIFNτ sequences.

The recombinant plasmid pBS24Ub-IFNτ was transformed into the yeast S.cerevisiae. Transformed yeast cells were cultured, lysed and therecombinant IFNτ (r-IFNτ) protein isolated from the cell lysates.

The amount of r-IFNτ was quantified by radioimmunoassay. Microsequencingof the purified r-IFNτ was carried out. The results demonstratedidentity with native OvIFNτ through the first 15 amino acids. Theresults also confirmed that the ubiquitin/IFNτ fusion protein wascorrectly processed in vivo.

Recombinant IFNτ obtained by this method exhibited antiviral activitysimilar to the antiviral activity of IFNτ purified fromconceptus-conditioned culture medium.

Other yeast vectors can be used in the practice of the presentinvention. They include 2 micron plasmid vectors (Ludwig, et al.), yeastintegrating plasmids (YIps; e.g., Shaw, et al.), YEP vectors (Shen, etal.), yeast centromere plasmids (YCps; e.g., Ernst), and the like.Preferably, the vectors include an expression cassette containing aneffective yeast promoter, such as the MFα1 promoter (Ernst, Bayne, etal.), GADPH promoter (glyceraldehyde-3-phosphate-dehydrogenase; Wu, etal.), the galactose-inducible GAL10 promoter (Ludwig, et al., Feher, etal., Shen, et al.), or the methanol-regulated alcohol oxidase (AOX)promoter (Tschopp, et al.). The AOX promoter is particularly useful inPichia pastoris host cells (for example, the AOX promoter is used inpHIL and pPIC vectors included in the Pichia expression kit, availablefrom Invitrogen, San Diego, Calif.).

The expression cassette may include additional elements to facilitateexpression and purification of the recombinant protein, and/or tofacilitate the insertion of the cassette into a vector or a yeastchromosome. For example, the cassette may include a signal sequence todirect secretion of the protein. An exemplary signal sequence suitablefor use in a variety of yeast expression vectors is the MFα1 pre-prosignal sequence (Bayne, et al., Ludwig, et al., Shaw, et al.). Othersignal sequences may also be used. For example, the Phol signal sequence(Elliot, et al.) is particularly effective in Pichia Pastoris andSchizosaccharomyces pombe host cells.

Exemplary expression cassettes include (i) a cassette containing (5' to3') the AOX promoter, the Pho1 signal sequence, and a DNA sequenceencoding OvIFNτ, for expression in P. pastoris host cells, and (ii) acassette containing (5' to 3') the MFα1 promoter, the MFα1 pre-prosignal sequence, and a DNA sequence encoding IFNτ, for expression in S.cerevisiae host cells.

Additional yeast vectors suitable for use with the present inventioninclude, but are not limited to, other vectors with regulatableexpression (Hitzeman, et al.; Rutter, et al.; Oeda, et al.). The yeasttransformation host is typically Saccharomyces cerevisiae, however, asillustrated above, other yeast suitable for transformation can be usedas well (e.g., Schizosaccharomyces pombe, Pichia pastoris and the like).

The DNA encoding the IFNτ polypeptide can be cloned into any number ofcommercially available vectors to generate expression of the polypeptidein the appropriate host system. These systems include the abovedescribed bacterial and yeast expression systems as well as thefollowing: baculovirus expression (Reilly, et al.; Beames, et al.;Clontech, Palo Alto, Calif.); plant cell expression, transgenic plantexpression (e.g., S.B. Gelvin and R.A. Schilperoot, Plant MolecularBiology, 1988), and expression in mammalian cells (Clontech, Palo Alto,Calif.; Gibco-BRL, Gaithersburg Md.). These recombinant polypeptides canbe expressed as fusion proteins or as native proteins. A number offeatures can be engineered into the expression vectors, such as leadersequences which promote the secretion of the expressed sequences intoculture medium. The recombinantly produced polypeptides are typicallyisolated from lysed cells or culture media. Purification can be carriedout by methods known in the art including salt fractionation, ionexchange chromatography, and affinity chromatography. Immunoaffinitychromatography can be employed, as described above, using antibodiesgenerated based on the IFNτ polypeptides.

B. Human Interferon-τ

1. Identification and Cloning of Human Genomic Sequences Encoding anInterferon-τ Protein.

Human genomic DNA was screened for sequences homologous to interferon-τ(Example 5). Several sequences that hybridized with the OvIFNτ cDNAprobe were identified. Several clones containing partial sequences ofhuman interferon-τ were then isolated (Example 6). Two synthetic 25-meroligonucleotides, corresponding to sequences from the OvIFNτ cDNA(Imakawa, et al., 1987; Whaley, et al., 1994) were synthesized. Theseprimers were employed in amplification reactions using DNA derived fromthe following two cDNA libraries: human placenta and humancytotrophoblast cells isolated from term placenta. The resultingamplified DNA fragments were electrophoretically separated and a bandcontaining human IFNτ amplification products was isolated. Theamplification products were subcloned and the inserted amplificationproducts sequenced using the dideoxy termination method.

Comparison of sequences from five of these clones revealed a high degreeof sequence homology between the isolates, but the sequences were notidentical. This result suggests the existence of multiple variants ofhuman interferon-τ genes. Analysis of the nucleotide and proteinsequences suggests that human interferon-τ genes may be classified onthe basis of sequence homology into at least three groups. The groupsare presented below.

Example 7 describes the isolation of several full-length human IFNτgenes. High molecular weight DNA was isolated from human peripheralblood mononuclear cells (PBMCs) and size-fractionated. Fractions weretested for the presence of IFNτ sequences using polymerase chainreaction: DNA molecules from fractions that tested amplificationpositive were used to generate a subgenomic library in λgt11.

This subgenomic library was plated and hybridized with an OvIFNτ cDNAprobe (Example 7A). Approximately 20 clones were identified thathybridized to the probe. Plaques corresponding to the positive cloneswere passaged, DNA isolated and analyzed by amplification reactionsusing OvIFNτ primers. Of these twenty plaques, six plaques generatedpositive PCR signals. The phage from these six clones were purified andthe inserts sequenced. One of the inserts from one of these six cloneswas used as a hybridization probe in the following screening.

Recombinant phage from the λgt11 subgenomic library were screened usingthe hybridization probe just described (Example 7B). Five clones givingpositive hybridization signals were isolated and the inserts sequenced.The sequences from three of the clones overlapped, and the resultingconsensus nucleic acid sequence (HuIFNτ1) is presented as SEQ ID NO:11with the predicted protein coding sequence presented as SEQ ID NO:12.The predicted mature protein coding sequence is presented as SEQ IDNO:4. The sequences from the other two clones are presented as SEQ IDNO:29 (HuIFNτ2) and SEQ ID NO:31 (HuIFNτ3). The predicted mature aminoacid sequence from HuIFNτ2 is presented as SEQ ID NO:30. The predictedamino acid sequence from HuIFNτ3 is presented as SEQ ID NO:32, and themature amino acid sequence as SEQ ID NO:34.

Comparison of the predicted protein sequences (FIG. 3) of one of thehuman interferon-τ genes (SEQ ID NO:4) and the ovine interferon-Υ genedemonstrates the levels of sequence homology and divergence at the aminoacid level.

An alignment of the nucleic acid sequences of the seven humaninterferon-τ nucleic acid sequences, described herein (Examples 6 and7), with ovine interferon-τ is shown in FIGS. 19A and 19B. Sequences ofOvIFNτ (oIFNτ), HuIFNτ1, HuIFNτ2, and HuIFNτ3 start at the upper leftcorner of FIG. 19A with the initiation ATG codon and continue throughthe second page of the figure. Sequences of HuIFNτ4, HuIFNτ5, HuIFNτ6and HuIFNτ7 start approximately half-way down FIG. 19A with the CAGcodon at amino acid position 40 (to the right of the exclamation marks)and continue through the second page of the figure. The 5' and 3' endsof each of the clones for HuIFNτ4, HuIFNτ5, HuIFNτ6 and HuIFNτ7 arerepresented by exclamation marks.

The complete coding sequence of OvIFNτ is presented in the top row ofeach aligned set. Nucleotides in the other sequences are indicated onlyat positions where they differ from those of OvIFNτ. Lower case lettersrepresent nucleotide changes that do not result in amino acid changes,while upper case letters represent those changes that result in an aminoacid substitution.

An alignment of the seven corresponding amino acid sequences,constructed in essentially the same manner as described above, ispresented in FIGS. 20A and 20B. As above, the complete amino acidsequence of OvIFNτ is presented in the top row, and amino acids of othersequences are indicated only at positions where they differ from theovine sequence.

An examination of the alignments reveals that the seven sequences may begrouped into at least three groups. Group I contains HuIFNτ1 andHuIFNτ2, group II contains HuIFNτ3, HuIFNτ4 and HuIFNτ5, and group IIIcontains HuIFNτ6 and HuIFNτ7. These groups may represent families ofinterferon-τ genes having distinct cellular functions.

These groupings were established based on the following criteria. Inmature proteins, Group I HuIFNτs have an asparagine (ASN) at amino acidposition number 95 (numbers in reference to FIGS. 20A to 20B), amethionine (MET) at amino acid position number 104, and a leucine (LEU)at amino acid position number 120; Group II HuIFNτs have an asparticacid (ASP) at amino acid position number 95, a threonine (THR) at aminoacid position number 104, and a methionine (MET) at amino acid positionnumber 120; and Group III HuIFNτs have an arginine (ARG) at amino acidposition number 72, a valine (VAL) at amino acid position number 120,and a serine (SER) at amino acid position number 122.

The nucleic acid and polypeptide human IFNτ sequences presented as SEQID NO:3, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21, SEQ IDNO:22, SEQ ID N0:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, and SEQ ID NO:34 can be used as the source forspecific primers and probes to detect isolates of further human IFNτcoding sequences and/or pseudogenes. Further, as described above, theremay be more than one isoform of the IFNτ protein and more than onecoding sequence per species. The specific nucleic acid probes used inthe practice of the present invention and antibodies reactive with theIFNτ polypeptides of the present invention may be useful to isolateunidentified variants of interferon-τ in mammals, according to themethods of the invention disclosed herein.

2. Characterization of the Expression of Interferon-τ in Human Tissues.

Human placental cDNA libraries and an ovine cDNA library were analyzedby hybridization to the OvIFNτ cDNA probe (Example 8). This DNAhybridization analysis suggested that the IFNτ-signals from human cDNAlibraries were approximately 1/100 of the signal obtained using theovine cDNA library. OvIFNτ cDNAs constitute around 0.4% of the ovinecDNA library. Accordingly, the abundance of human cDNAs responding tothe OvIFNτ probe appears to be low, at least in the term placenta fromwhich the cDNA libraries were generated.

The presence of HuIFNτ mRNA in human term placenta and amniocytes werealso analyzed. The results suggest the presence of HuIFNτ mRNA in thefeto-placental annex. The aminocytes also expressed the messagescorresponding to OvIFNτ primers and human probe, suggesting that theexpression of IFNτ mRNA is not limited to the term placenta.

In addition, a RT-PCR analysis for the presence of HuIFNτ was applied tothe total cellular RNA isolated from human adult lymphocytes: theresults suggest that IFNτ mRNA exists in lymphocytes.

The expression of interferon-τ in human tissue was also examined usingin situ hybridization (Example 9). Sections from four healthy, differentterm and first trimester human placentas were examined. This analysisemployed a cDNA probe derived from the OvIFNτ cDNA sequences (Example9B). In situ hybridization was performed using an anti-sense RNA probe.In three separate experiments, specific hybridization was observed inall term and first trimester placental tissues.

First trimester placental villi (composed of an outer layer ofsyncytiotrophoblast, an underlying layer of cytotrophoblast, and acentral stromal region with various types of mesenchymal cells)displayed the highest transcript level of IFNτ in the cytotrophoblastcells. Less intense but detectable levels were present in both thesyncytiotrophoblast and stromal cells. A similar pattern of transcriptexpression was demonstrated in the placental villi of term tissue butthe level of signal detection was low. First trimester extravilloustrophoblasts displayed the highest amount of message and stainedpositive when present in the maternal blood spaces within the decidua.

Howatson, et al., (1988) noted IFNα production in thesyncytiotrophoblast of chorionic villi in both first trimester and termtissues. Also, Paulesu, et al. (1991) observed IFNα in extravilloustrophoblast as well as syncytiotrophoblasts, noting more intense andabundant reactivity in first trimester placental tissue when compared tothose taken at term. These investigators employed antibodies raisedagainst human IFNα subtypes, and none observed any IFNα in the villouscytotrophoblasts.

The present results demonstrate that the human IFNτ gene is highlyexpressed in early placental tissues by migrating extravilloustrophoblasts, but is also expressed in villous syncytiotrophoblasts,cytotrophoblasts, and various stromal cells. These results demonstratethe detection of IFNτ transcripts in human pregnancy tissues, and IFNτexpression in the villous cytotrophoblasts as well as the extravilloustrophoblast of first trimester placenta.

C. Antiviral Properties of Interferon-τ.

The antiviral activity of OvIFNτ has been evaluated against a number ofviruses, including both RNA and DNA viruses. The relative specificactivity of OvIFNτ, purified to homogeneity, was evaluated in antiviralassays (Example 10). OvIFNτ had a higher specific antiviral activitythan either rBoIFNα or rBoIFNγ (Example 10, Table 3).

One advantage of the present invention is that OvIFNτ has potentantiviral activity with limited cytotoxic effects. Highly purifiedOvIFNτ was tested for anti-retroviral and cytotoxic effects onperipheral blood lymphocytes exposed to feline AIDS and human AIDSretroviruses (Bazer, F. W., et al., 1989). The feline AIDS lentivirusproduces a chronic AIDS-like syndrome in cats and is a model for humanAIDS (Pederson, et al., 1987). Replication of either virus in peripheralblood lymphocytes (PBL) was monitored by reverse transcriptase (RT)activity in culture supernatants over time.

To determine IFNτ antiviral activity against FIV and HIV, RNA-dependentDNA polymerase RT activity was assayed in FIV- and HIV-infected felineand human PBL cultures treated with OvIFNτ (Example 11). Replication ofFIV was reduced to about one-third of control values when cells werecultured in the presence of OvIFNτ. Addition of OvIFNτ produced a rapid,dose-dependent decrease in reverse transcriptase (RT) activity (Example11, Table 4). While concentrations as low as 0.62 ng/ml of IFNτinhibited viral replication, much higher concentrations (40 ng/ml)having greater effects on RT-activity were without toxic effects on thecells. The results suggest that replication of the felineimmunodeficiency virus was reduced significantly compared to controlvalues when cells were cultured in the presence of OvIFNτ.

IFNτ appeared to exert no cytotoxic effect on the cells hosting theretrovirus. This was true even when IFNτ was present at 40 ng per ml ofculture medium. This concentration of IFNτ is equivalent to about 8,000antiviral units of alpha interferon--when OvIFNτ and HuIFNα are eachassayed for their ability to protect Madin-Darby bovine kidney cellsfrom lysis by vesicular stomatitis virus (lysis assay as described byPontzer, et al., 1988).

IFNτ was also tested for activity against HIV replication in humancells. Human peripheral lymphocytes, which had been infected with HIVwere treated with varying concentrations of IFNτ (Example 12).Replication of HIV in peripheral blood lymphocytes was monitored byreverse transcriptase activity in culture supernatants over time. Over arange of concentrations of IFNτ produced significant anti-HIV effects(Example 12, Table 5). A concentration of only 10 ng/ml resulted in overa 50% reduction in RT activity after only six days. A concentration of500 ng/ml resulted in a 90% reduction in RT activity within 10 days.Further, there was no evidence of any cytotoxic effects attributable tothe administration of IFNτ (Example 12, Table 5).

Further, the antiviral effects of IFNτ against HIV were evaluated bytreating human PBMC cells with various amounts of either recombinantIFNτ or recombinant human IFNα at the time of infection with HIV(Example 18). The data from these experiments (Example 18, Table 11)support the conclusion that, at similar concentrations, IFNα and IFNτare effective in reducing the replication of HIV in human lymphocytes.However, treatment of cells with IFNα resulted in cytotoxicity, whereasno such cytotoxicity was observed with treatment using IFNτ, even whenIFNτ was used at much higher concentrations. No cytotoxicity wasobserved using IFNτ, even when IFNτwas used at 200 times the dosage ofinterferon-alpha II.

Both FIV and HIV reverse transcriptase themselves were unaffected byIFNτ in the absence of PBL. Therefore, the antiviral activity is not dueto a direct effect on the viral RT.

Interferon-τ has also been shown to inhibit Hepatitis B Virus DNAreplication in hepatocytes (Example 18). A human cell line derived fromliver cells transfected with Hepatitis B Virus (HBV) was used to testthe antiviral effects of IFNτ. The cells were treated with both the IFNαand IFNτ over a range of concentrations. Both IFNα and IFNτ reduced DNAproduction by approximately two-fold compared to the no interferoncontrol.

To demonstrate that the effect of the interferons was specific to theinfecting virus and not the result of an effect on general cellmetabolism, the hepatocytes were examined for the effects of IFNα andIFNτ on hepatospecific mRNA production (Example 18). Two hepatocytespecific proteins, Apo E and Apo A1, were detected by hybridizationanalysis. There was no apparent reduction of mRNA production for eitherhepatospecific mRNA at concentrations up to 40,000 units/ml of eitherIFNα or IFNτ. Further, no evidence for hepatotoxicity with IFNτ was seenin this assay.

The effects of recombinant ovine interferon tau (roIFNτ) on ovinelentivirus replication (OvLV) were also evaluated. In vitro effects wereassayed by infecting goat synovial membrane cells with serial dilutionsof OvLV. The infected cells were treated daily with roIFNτ (0-2, 500antiviral units/ml AVU/ml!) for 6 to 12 days, and virus replication andcytopathic effect (CPE; e.g., as in Example 2) were evaluated.

Evaluation methods included viral growth curves, cell proliferationassay (e.g., as in Examples 13, 14 or 15), syncytia formation assay(e.g., as in Nagy, et al., Dalgleish, et al.), and quantitation ofproviral DNA by PCR and reverse transcriptase assay (Mullis, Mullis, etal.). A reduction (p<0.001) of OvLV titer and CPE (80-99%) was observedin the roIFNτ-treated cultures.

In vivo effects of roIFNτ were assayed by inoculating twenty-fournewborn lambs with 5×10⁶ TCID₅₀ of OvLV strain 85/34. Eleven of theselambs were treated with 10⁵ -10⁶ AVU/Kg of roIFNτ once a day for 30 dayspost-inoculation (PI) and twice a week thereafter. Thirteen lambs wereused as controls. Virus titers in blood, as determined by an end pointdilution method, peaked at 4-6 weeks PI in both groups. OvLV titers inroIFNτ-treated lambs were reduced relative to control animals. Thelargest reduction, a 90% decrease in OvLV titer in treated animalsrelative to control animals (p<0.01), was obtained 4 weeks PI.

The OvLV studies described above indicate that recombinant ovIFNτ cansignificantly reduce OvLV replication, and suggest that IFNτ may be usedto control clinical diseases caused by lentivirus infections. Takentogether with the other antiviral data, these results suggest that IFNτis an effective antiviral agent against a wide variety of viruses,including both RNA and DNA viruses.

Ovine interferon-τ may be useful in veterinary applications including,but not limited to, the treatment of the following viral diseases:feline leukemia virus, ovine progressive pneumonia virus, ovinelentivirus, equine infectious anemia virus, bovine immunodeficiencyvirus, visna-maedi virus, and caprine arthritis encephalitis.

Human interferon-τ may be used for the treatment of, for example, thefollowing viral diseases: human immunodeficiency virus (HIV), hepatitisc virus (HCV) and hepatitis B virus (HBV).

D. Antiproliferative Properties of IFNτ

The effects of IFNτ on cellular growth have also been examined. In oneanalysis, anti-cellular growth activity was examined using a colonyinhibition assay (Example 13). Human amnion (WISH) or MDBK cells wereplated at low cell densities to form colonies originating from singlecells. Dilutions of interferons were added to triplicate wells and theplates were incubated to allow colony formation. IFNτ inhibited bothcolony size and number in these assays. IFNτ was more effective atinhibiting cell proliferation of the human cell line (WISH) than humanIFNα. The antiproliferative activity of IFNτ was dose-dependent. Highconcentrations of IFNτ stopped proliferation, while cell viability wasnot impaired.

Based on cell cycle analysis, using flow cytometry, IFNτ appears toinhibit progress of cells through S phase. These results demonstrate theantiproliferative effect of IFNτ, and underscore its low cytotoxicity.

The antiproliferative effects of IFNτ were also studied for rat andbovine cell lines (Example 14). The rate of ³ H-thymidine incorporationwas used to assess the rate of cellular proliferation. The data obtaineddemonstrate that IFNτ drastically reduced the rate of cellularproliferation (Example 14, Table 7) for each tested cell line.

The antiproliferative activity and lack of toxicity of IFNτ was furtherexamined using a series of human tumor cell lines (Example 15). Avariety of human tumor cell lines were selected from the standard linesused in NIH screening procedure for antineoplastic agents (Pontzer, C.H., et al., (1991)). At least one cell line from each major neoplasticcategory was examined.

The following cell lines were obtained from American Type CultureCollection (12301 Parklawn Dr., Rockville Md. 20852):

    ______________________________________    NCI-H460     human lung large cell carcinoma;    DLD-1        human colon adenocarcinoma;    SK-MEL-28    human malignant melanoma;    ACHN         human renal adenocarcinoma;    HL-60        human promyelocytic leukemia;    H9           human T cell lymphoma;    HUT 78       human cutaneous T cell lymphoma; and    MCF7         human breast adenocarcinoma.    ______________________________________

As above, the antiproliferative activity was evaluated by measuring therate of ³ H-thymidine incorporation into cells which have been treatedwith IFNτ. Significant differences between treatments were assessed byan analysis of variance followed by Scheffe's F-test. Cell cycleanalysis was performed by flow cytometry.

Examination of IFNτ inhibition of MCF7 (breast adenocarcinoma)proliferation demonstrated that IFNτ reduced MCF7 proliferation in adose-dependent manner. A 50% reduction in ³ H-thymidine was observedwith 10,000 units/ml of IFNτ (Example 15, Table 8). This cell line hadpreviously been found to be unresponsive to anti-estrogen treatment.

A comparison of the antiproliferative effects of IFNτ and IFNα wasconducted using HL-60 (human promyelocytic leukemia) cells. Results withthe promyelocytic leukemia HL-60 are typical of those obtained comparingIFNτ with human IFNα (Example 15). Concentrations as low as 100 units/mlof both IFNs produced significant (>60%) growth reduction. Increasingamounts of IFNs further decreased tumor cell proliferation (FIG. 4).High doses of HuIFNα, but not OvIFNτ, were cytotoxic (FIG. 5). Cellviability was reduced by approximately 80% by IFNα. By contrast, nearly100% of the IFNτ-treated cells remained viable when IFNτ was applied at10,000 units/ml. Thus, while both interferons inhibit proliferation,only IFNτ is without cytotoxicity. This lack of toxicity provides anadvantage of IFNτ for use in vivo therapies.

The human cutaneous T cell lymphoma, HUT 78, responded similarly toHL-60 when treated with IFNτ (Example 15, FIG. 9). Both OvIFNτ andrHuIFNα reduce HUT 78 cell growth, but IFNα demonstrated adverse effectson cell viability.

The T cell lymphoma H9 was less sensitive to the antiproliferativeeffects of IFNα than the tumor cell lines described above. While IFNαwas not toxic to the H9 cells, it failed to inhibit cell divisionsignificantly at any of the concentrations examined (Example 15, FIG.10). In contrast, IFNτ was observed to reduce H9 growth by approximately60%. Thus, only OvIFNτ is an effective growth inhibitor of this T celllymphoma.

In three additional tumor cell lines (NCI-H460, DLD-1 and SK-MEL-28)IFNτ and IFNα were equally efficacious antitumor agents. In themelanoma, SK-MEL28, inhibition of proliferation by IFNα was accomplishedby a 13% drop in viability, while IFNτ was not cytotoxic. In themajority of tumors examined, IFNτ is equal or preferable to IFNα as anantineoplastic agent against human tumors.

IFNτ exhibits antiproliferative activity against human tumor cellswithout toxicity and is as potent or more potent than human IFNα.Clinical trials of the IFNα2s have shown them to be effective antitumoragents (Dianzani, F., 1992; Krown, 1987). One advantage of IFNτ as atherapeutic is the elimination of toxic effects seen with high dosesIFNαs.

An additional application of the IFNτ is against tumors like Kaposi'ssarcoma (associated with HIV infection) where the antineoplastic effectsof IFNτ are coupled with IFNτ ability to inhibit retroviral growth.

The in vivo efficacy of interferon-τ treatment was examined in a mousesystem (Example 16). B16-F10 is a syngeneic mouse transplantable tumorselected because of its high incidence of pulmonary metastases (Poste,et al., 1981). Interferon treatment was initiated 3 days after theintroduction of the tumor cells. The in vivo administration of IFNτdramatically reduced B16-F10 pulmonary tumors. Thus, IFNτ appears to bean efficacious antineoplastic agent in vivo as well as in vitro.

These results support the usefulness of human IFNτ for use in methods toinhibit or reduced tumor cell growth, including, but are not limited to,the following types of tumor cells: human carcinoma cells, hematopoieticcancer cells, human leukemia cells, human lymphoma cells, human melanomacells and steroid-sensitive tumor cells (for example, mammary tumorcells).

E. Cytotoxicity of Interferons

One advantage of IFNτ over other interferons, such as IFNα, is thattreatment of a subject with therapeutic doses of IFNτ does not appear tobe associated with cytotoxicity. In particular, IFN-τ appears to benon-toxic at concentrations at which IFN-β induces toxicity. This isdemonstrated by experiments in which L929 cells were treated withvarious concentrations of either oIFNτ or MuIFN-β (Lee Biomolecular, SanDiego, Calif.), ranging from 6000 U/ml to 200,000 U/ml (Example 18E).

oIFNτ, MuIFN-β or medium (control) were added at time zero and the cellswere incubated for 72 hours. The results of the experiments arepresented in FIG. 21. The percent of live cells (relative to control) isindicated along the y-axis (± standard error). One hundred percent isequal to the viability of L929 cells treated with medium alone. Theresults indicate that oIFNτ is essentially non-toxic at concentrationsup to 100,000 U/ml, and is significantly less toxic than MuIFN-β overthe entire therapeutic range of the compounds.

It has been previously demonstrated that in vivo treatment with both ofthe type I IFNs, IFNβ and IFNα in humans and animals causes toxicitymanifested as a number of side effects including fever, lethargy,tachycardia, weight loss, and leukopenia (Degre, 1974; Fent and Zbinden,1987). The effect of in vivo treatment with IFNτ, IFNβ and IFNα (10⁵U/injection) on total white blood cell (WBC), total lymphocyte countsand weight measurements in NZW mice (Table 13) was examined as describedin Example 18F. No significant difference between IFNτ treated anduntreated mice was observed for WBC, lymphocyte counts or weight change.

In comparison, IFNβ treated mice exhibited a 31.7% depression inlymphocyte counts 12 hours after injection. Further, depression oflymphocyte counts continued 24 hours after IFNβ injection. IFNα treatedmice exhibited a 55.8% lymphocyte depression and significant weight loss12 hours after injection. Thus, IFNτ appears to lack toxicity in vivounlike IFNβ and IFNα as evidenced by studies of peripheral blood andweight measurements.

oIFNτ is 172 amino acids long compared to 165 or 166 amino acids forIFNα. The carboxyl-terminal portion of oIFNτ is hydrophilic and thoughtto be surface accessible. To explore whether this carboxyl "tail"interacts with the binding epitope of oIFNτ to mediate the relative lackof cytotoxicity, a series of deletion mutants were generated.

The carboxyl terminal 2, 6 and 10 amino acids of oIFNτ were removed bycassette mutagenesis of a synthetic oIFNτ gene. The mutant (variant)synthetic genes were cloned into the pHIL-S1 Pichia expression plasmid(Invitrogen, San Diego, Calif.), the plasmid was cut with BglII, and thelinearized plasmid was used to transform Pichia pastoris (strain GS115;Invitrogen) spheroplasts according to the manufacturer's instructions.

Recombinant variant proteins expressed by transformed His⁺ Mut⁻ yeastcells were purified and used to determine in vitro antiviral activityand relative cytotoxicity of the variants compared to intact oIFNτ andIFN-α. The cytoxicity of the variants was distributed between the thatof oIFNτ and IFN-α. Variants with shorter deletions were more similar intheir cytotoxic properties to oIFNτ, while those with longer deletionswere more similar to IFN-α.

While not wishing to be bound by any specific molecular mechanismsunderlying the properties of IFNτ, the results of the experimentssuggest that the C-terminal 10 amino acids of IFNτ may play a role inthe decreased cytotoxicity of IFNτ relative to other interferons.

III. Interferon-τ Polypeptide Fragments, Protein Modeling and ProteinModifications

A. IFNτ Polypeptide Fragments

The variety of IFNτ activities, its potency and lack of cytotoxicity, astaught by the present specification, suggest the importance ofstructure/function analysis for this novel interferon. The structuralbasis for OvIFNτ function has been examined using six overlappingsynthetic peptides corresponding to the entire OvIFNτ sequence (FIG. 6).The corresponding polypeptides derived from the ovine IFNτ sequence arepresented as SEQ ID NO:5 to SEQ ID NO:10. Three peptides representingamino acids 1-37, 62-92 and 139-172 have been shown to inhibit IFNτantiviral activity (Example 17). The peptides were effective competitorsat concentrations of 300 μM and above.

The synthetic polypeptide representing the C-terminal region of ovIFNτ,OvIFNτ (139-172), and the internal peptide OvIFNτ (62-92), inhibitedIFNτ and rBoIFNα_(II) antiviral activity to the same extent, while theN-terminal peptide OvIFNτ (1-37) was more effective in inhibiting OvIFNτantiviral activity. Dose-response data indicated that IFNτ (62-92) andIFNτ (139-172) inhibited IFNτ antiviral activity to similar extents. Thesame peptides that blocked IFNτ antiviral activity also blocked theantiviral activity of recombinant bovine IFNα (rBoIFNα); recombinantbovine IFNγ was unaffected by the peptides. These two IFNτ peptides mayrepresent common receptor binding regions for IFNτ and various IFNαs.

The two synthetic peptides OvIFNτ (1-37) and OvIFNτ (139-172) alsoblocked OvIFNτ anti-FIV and anti-HIV activity (Example 17; FIGS. 11A and11B). While both peptides blocked FIV RT activity, only the C-terminalpeptide, OvIFNτ (139-172), appeared to be an efficient inhibitor ofvesicular stomatitis virus activity on the feline cell line, Fc9.

The above data taken together suggest that the C-terminal regions oftype I interferons may bind to common site on the type I interferonreceptor, while the N-terminal region may be involved in the elicitationof unique functions. These results suggest that portions of the IFNτmolecule may be used to substitute regions of interferon alphamolecules. For example, the region of an interferon alpha molecule thatis responsible for increased cytotoxicity, relative to IFNτ treatment,can be identified by substituting polypeptide regions derived from IFNτfor regions of an interferon alpha molecule. Such substitutions can becarried out by manipulation of synthetic genes (see below) encoding theselected IFNτ and interferon alpha molecules, coupled to the functionalassays described herein (such as, antiviral, antiproliferative andcytoxicity assays).

Polyclonal anti-peptide antisera against the IFNτ peptides yieldedsimilar results as the polypeptide inhibition studies, described above.Antibodies directed against the same three regions (OvIFNτ (1-37), IFNτ(62-92) and IFNτ (139-172)) blocked OvIFNτ function, confirming theimportance of these three domains in antiviral activity (Example 17).These peptides, although apparently binding to the interferon receptor,did not in and of themselves elicit interferon-like effects in thecells.

The antiproliferative activity of IFNτ (Example 17, Table 11) involved afurther region of the molecule, since IFNτ (119-150) was the mosteffective inhibitor of OvIFNτ-induced reduction of cell proliferation.This results suggests that the region of the molecule primarilyresponsible for inhibition of cell growth is the IFNτ (119-150) region.This region of the IFNτ molecule may be useful alone or fused to otherproteins (such as serum albumin, an antibody or an interferon alphapolypeptide) as an antineoplastic agent. A conjugated protein between anN-terminal peptide derived from human interferon-α and serum albumin wasshown to have anticellular proliferation activity (Ruegg, et al., 1990).

Finally, binding of ¹²⁵ I-OvIFNτ to its receptor on MDBK cells could beblocked by antisera to 4 of the 6 peptides; the 4 polypeptidesrepresenting amino acids 1-37, 62-92, 119-150 and 139-172 of OvIFNτ.This reflects the multiple binding domains as well as the functionalsignificance of these regions. Since different regions of IFNτ areinvolved in elicitation of different functions, modification of selectedamino acids could potentially result in IFNτ-like interferons withselective biological activity.

Polypeptide fragments of human IFNτ proteins, having similar propertiesto the OvIFNτ polypeptides just described, are proposed based on thedata presented above for OvIFNτ polypeptide fragments combined with theHuIFNτ sequence information disclosed herein. Such human-sequencederived polypeptides include, but are not limited to, the following: SEQID NO:15 to SEQ ID NO:20, and SEQ ID NO:35 to SEQ ID NO:40.

The above data demonstrate the identification of synthetic peptideshaving four discontinuous sites on the OvIFNτ protein that are involvedin receptor interaction and biological activity. In order to elucidatethe structural relationship of these regions, modeling of the threedimensional structure of IFNτ was undertaken. A three dimensional modelwould be useful in interpretation of existing data and the design offuture structure/function studies.

B. Molecular Modeling

Combining circular dichroism (CD) data of both the full lengthrecombinant OvIFNτ and IFNβ (a protein of known three dimensionalstructure (Senda, et al., 1992)), a model of OvIFNτ was constructed. Themost striking feature of this model is that IFNτ falls into a class ofproteins with a four-helix bundle motif. The CD spectra of IFNτ wastaken on an AVIV 60 S spectropolarimeter. Two different methods wereemployed for secondary structure estimations, the algorithm of Perczel,et al., (1991) and variable selection by W. C. Johnson, Jr. (1992).

Secondary structure estimations of the spectra indicate 70-75% alphahelix (characterized by minima at 222 and 208 nm and maximum at 190 nm).The variable selection algorithm estimates the remainder of the moleculeto be 20% beta sheet and 10% turn. The Chang method estimates theremainder to be 30% random coil. Alignment of IFNτ and IFNβ sequencesrevealed homology between the two molecules, specifically in the regionsof known helical structure in IFNβ. Sequence analysis of IFNτ alsoshowed that proposed helical regions possess an apolar periodicityindicative of a four-helix bundle motif.

The final modeling step was to apply the IFNβ x-ray crystallographiccoordinates of the IFNβ carbon backbone to the IFNτ sequence. Thefunctionally active domains of IFNτ, identified above, were localized toone side of the molecule and found to be in close spatial proximity.This is consistent with multiple binding sites on IFNτ interactingsimultaneously with the type I IFN receptor.

The three dimensional modeling data coupled with the function datadescribed above, provides the information necessary to introducesequence variations into specific regions of IFNτ to enhance selectedfunctions (e.g., antiviral or anticellular proliferation) or tosubstitute a region(s) of selected function into other interferonmolecules (e.g., antiviral, antineoplastic, or reduced cytotoxicity).

C. Recombinant and Synthetic Manipulations

The construction of a synthetic gene for OvIFNτ is described in Example3. Briefly, an amino acid sequence of ovIFNτ was back-translated from anovIFNτ cDNA (Imakawa, et al., 1987) using optimal codon usage for E.coli. The sequence was edited to include 20, unique, restriction sitesspaced throughout the length of the construct. This 540 base pairsynthetic gene sequence was divided into 11 oligonucleotide fragments.Individual fragments were synthesized and cloned, either single ordouble stranded, into either pTZ 19R, pTZ 18R or pBluescript, amplifiedand fused. The synthetic OvIFNτ construct was then cloned into amodified pIN-III-ompA expression vector for expression in bacteria andalso cloned into a yeast expression plasmid. A similarly constructedhuman IFNτ synthetic gene (SEQ ID NO:3) has been designed, constructedand expressed in yeast cells.

Expression of the OvIFNτ synthetic gene in yeast (Example 4) allowedover production of recombinant IFNτ in S. cerevisiae: large quantities(5-20 mg/l) of recombinant IFNτ can be purified from soluble yeastextract using sequential ion exchange and molecular sievechromatography. Recombinant IFNτ purified in this fashion exhibitedpotent antiviral activity (2 to 3×10⁸ units/mg) similar to nativeOvIFNτ.

The synthetic gene construct facilitates introduction of mutations forpossible enhancement of antitumor (anticellular proliferative) andantiviral activities. Further, the disparate regions of the moleculeresponsible for different functions can be modified independently togenerate a molecule with a desired function. For example, two deletionmutants, OvIFNτ (1-162) and OvIFNτ(1-166), have been constructed toexamine the role of carboxy terminal sequences in IFNτ molecules.

Additional mutant IFNτ molecules have been constructed to identifyresidues critical for antiproliferative activity. For example, oneparticular residue, TYR 123 has been implicated in the anticellularproliferative activity of IFNα (McInnes, et al., 1989). The equivalentof TYR 123 in IFNτ is contained within peptide OvIFNτ (119-150): thispolypeptide inhibits OvIFNτ and human IFNα antiproliferative activity.Mutations converting TYR 123 to conservative (TRP) and nonconservative(ASP) substitutions have been generated, as well as mutant sequenceshaving deletion of this residue. The codon for TYR 123 is located withinan SspI site; elimination of this site has been used for screening. Theantiproliferative activity of these mutant IFNτ is evaluated asdescribed herein.

Synthetic peptides can be generated which correspond to the IFNτpolypeptides of the present invention. Synthetic peptides can becommercially synthesized or prepared using standard methods andapparatus in the art (Applied Biosystems, Foster City Calif.).

Alternatively, oligonucleotide sequences encoding peptides can be eithersynthesized directly by standard methods of oligonucleotide synthesis,or, in the case of large coding sequences, synthesized by a series ofcloning steps involving a tandem array of multiple oligonucleotidefragments corresponding to the coding sequence (Crea; Yoshio et al.;Eaton et al.). oligonucleotide coding sequences can be expressed bystandard recombinant procedures (Maniatis et al.; Ausubel et al.).

The biological activities of the interferon-τ polypeptides describedabove can be exploited using either the interferon-τ polypeptides aloneor conjugated with other proteins (see below).

IV. Production of Fusion Proteins

In another aspect, the present invention includes interferon-τ orinterferon-τ -derived polypeptides covalently attached to a secondpolypeptide to form a fused, or hybrid, protein. The interferon-τsequences making up such fused proteins can be recombinantly producedinterferon-τ or a bioactive portion thereof, as described above.

For example, where interferon-τ is used to inhibit viral expression,polypeptides derived from IFNτ demonstrating antiviral activity may beadvantageously fused with a soluble peptide, such as, serum albumin, anantibody (e.g., specific against an virus-specific cell surfaceantigen), or an interferon alpha polypeptide. In one embodiment, theIFNτ polypeptides provide a method of reducing the toxicity of otherinterferon molecules (e.g., IFNβ or IFNα) by replacingtoxicity-associated regions of such interferons with, for example,corresponding interferon-τ regions having lower toxicity. In anotherembodiment, fusion proteins are generated containing interferon-τregions that have anticellular proliferation properties. Such regionsmay be obtained from, for example, the human interferon-τ sequencesdisclosed herein.

The fused proteins of the present invention may be formed by chemicalconjugation or by recombinant techniques. In the former method, theinterferon-τ and second selected polypeptide are modified byconventional coupling agents for covalent attachment. In one exemplarymethod for coupling soluble serum albumin to an interferon-τpolypeptide, serum albumin is derivatized with N-succinimidyl-S-acetylthioacetate (Duncan), yielding thiolated serum albumin. The activatedserum albumin polypeptide is then reacted with interferon-τ derivatizedwith N-succinimidyl 3-(2-pyridyldithio) propionate (Cumber), to producethe fused protein joined through a disulfide linkage.

As an alternative method, recombinant interferon-τ may be prepared witha cysteine residue to allow disulfide coupling of the interferon-τ to anactivated ligand, thus simplifying the coupling reaction. Aninterferon-τ expression vector, used for production of recombinantinterferon-τ, can be modified for insertion of an internal or a terminalcysteine codon according to standard methods of site-directedmutagenesis (Ausubel, et al.).

In one method, a fused protein is prepared recombinantly using anexpression vector in which the coding sequence of a second selectedpolypeptide is joined to the interferon-τ coding sequence. For example,human serum albumin coding sequences can be fused in-frame to the codingsequence of an interferon-τ polypeptide, such as, SEQ ID NO:9, SEQ IDNO:19 or SEQ ID NO:39. The fused protein is then expressed using asuitable host cell. The fusion protein may be purified bymolecular-sieve and ion-exchange chromatography methods, with additionalpurification by polyacrylamide gel electrophoretic separation and/orHPLC chromatography, if necessary.

It will be appreciated from the above how interferon-τ-containing fusionproteins may be prepared. One variation on the above fusion is toexchange positions of the interferon-τ and selected second proteinmolecules in the fusion protein (e.g., carboxy terminal versus aminoterminal fusions). Further, internal portions of a native interferon-τpolypeptide (for example, amino acid regions of between 15 and 172 aminoacids) can be assembled into polypeptides where two or more suchinterferon-τ portions are contiguous that are normally discontinuous inthe native protein.

In addition to the above-described fusion proteins, the presentinvention also contemplates polypeptide compositions having (a) a humaninterferon-τ polypeptide, where said polypeptide is (i) derived from aninterferon-τ amino acid coding sequence, and (ii) between 15 and 172amino acids long, and (b) a second soluble polypeptide. Interferon-α andinterferon-β are examples of such second soluble polypeptides. IFNτpolypeptides associated with reduced toxicity may be co-administeredwith more toxic interferons to reduce the toxicity of the more toxicinterferons when used in pharmaceutical formulations or in therapeuticapplications. Such IFNτ polypeptides would, for example, reduce thetoxicity of IFNα but not interfere with IFNα antiviral properties.

V. Antibodies Reactive with Interferon-τ

Fusion proteins containing the polypeptide antigens of the presentinvention fused with the glutathione-S-transferase (Sj26) protein can beexpressed using the pGEX-GLI vector system in E. coli JM101 cells. Thefused Sj26 protein can be isolated readily by glutathione substrateaffinity chromatography (Smith). Expression and partial purification ofIFNτ proteins is described in (Example 20), and is applicable to any ofthe other soluble, induced polypeptides coded by sequences described bythe present invention.

Insoluble GST (sj26) fusion proteins can be purified by preparative gelelectrophoresis.

Alternatively, IFNTτ-β-galactosidase fusion proteins can be isolated asdescribed in Example 19.

Also included in the invention is an expression vector, such as thelambda gt11 or pGEX vectors described above, containing IFNτ codingsequences and expression control elements which allow expression of thecoding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector.

The DNA encoding the desired polypeptide can be cloned into any numberof vectors (discussed above) to generate expression of the polypeptidein the appropriate host system. These recombinant polypeptides can beexpressed as fusion proteins or as native proteins. A number of featurescan be engineered into the expression vectors, such as leader sequenceswhich promote the secretion of the expressed sequences into culturemedium. Recombinantly produced IFNτ, and polypeptides derived therefrom,are typically isolated from lysed cells or culture media. Purificationcan be carried out by methods known in the art including saltfractionation, ion exchange chromatography, and affinity chromatography.Immunoaffinity chromatography can be employed using antibodies generatedagainst selected IFNτ antigens.

In another aspect, the invention includes specific antibodies directedagainst the polypeptides of the present invention. Typically, to prepareantibodies, a host animal, such as a rabbit, is immunized with thepurified antigen or fused protein antigen. Hybrid, or fused, proteinsmay be generated using a variety of coding sequences derived from otherproteins, such as β-galactosidase or glutathione-S-transferase. The hostserum or plasma is collected following an appropriate time interval, andthis serum is tested for antibodies specific against the antigen.Example 20 describes the production of rabbit serum antibodies which arespecific against the IFNτ antigens in a Sj26/IFNτ hybrid protein. Thesetechniques can be applied to the all of the IFNτ molecules andpolypeptides derived therefrom.

The gamma globulin fraction or the IgG antibodies of immunized animalscan be obtained, for example, by use of saturated ammonium sulfate orDEAE Sephadex, or other techniques known to those skilled in the art forproducing polyclonal antibodies.

Alternatively, purified protein or fused protein may be used forproducing monoclonal antibodies. Here the spleen or lymphocytes from aanimal immunized with the selected polypeptide antigen are removed andimmortalized or used to prepare hybridomas by methods known to thoseskilled in the art (Harlow, et al.). Lymphocytes can be isolated from aperipheral blood sample. Epstein-Barr virus (EBV) can be used toimmortalize human lymphocytes or a fusion partner can be used to producehybridomas.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete antibodies of the desired specificity, forexample, by using the ELISA or Western blot method (Ausubel et al.).Experiments performed in support of the present invention have yieldedfour hybridomas producing monoclonal antibodies specific for ovine IFNτhave been isolated.

Antigenic regions of polypeptides are generally relatively small,typically 7 to 10 amino acids in length. Smaller fragments have beenidentified as antigenic regions. Interferon-τ polypeptide antigens areidentified as described above. The resulting DNA coding regions can beexpressed recombinantly either as fusion proteins or isolatedpolypeptides.

In addition, some amino acid sequences can be conveniently chemicallysynthesized (Applied Biosystems, Foster City, Calif.). Antigens obtainedby any of these methods may be directly used for the generation ofantibodies or they may be coupled to appropriate carrier molecules. Manysuch carriers are known in the art and are commercially available (e.g.,Pierce, Rockford Ill.).

Antibodies reactive with IFNτ are useful, for example, in the analysisof structure/function relationships.

VI. Utility

A. Reproductive

Although IFNτ bears some similarity to the IFNα family based onstructure and its potent antiviral properties, the IFNαs do not possessthe reproductive properties associated with IFNτ. For example,recombinant human IFNα had no effect on interestrous interval comparedto IFNτ, even when administered at twice the dose (Davis, et al., 1992).

Therefore, although IFNτ has some structural similarities to otherinterferons, it has very distinctive properties of its own: for example,the capability of significantly influencing the biochemical events ofthe estrous cycle.

The human IFNτ of the present invention can be used in methods ofenhancing fertility and prolonging the life span of the corpus luteum infemale mammals as generally described in Hansen, et al., hereinincorporated by reference. Further, the human interferon-τ of thepresent invention could be used to regulate growth and development ofuterine and/or fetal-placental tissues. The human IFNτ is particularlyuseful for treatment of humans, since potential antigenic responses areless likely using such a same-species protein.

B. Antiviral Properties

The antiviral activity of IFNτ has broad therapeutic applicationswithout the toxic effects that are usually associated with IFNαs.Although the presence of IFNτ in culture medium inhibited reversetranscriptase activity of the feline immunodeficiency virus (Example11), this is not due to a direct effect of IFNτ on the reversetranscriptase. Rather, IFNτ appears to induce the host cell to produce afactor(s) which is inhibitory to the reverse transcriptase of the virus.

IFNτ was found to exert its antiviral activity without adverse effectson the cells: no evidence of cytotoxic effects attributable to theadministration of IFNτ was observed. It is the lack of cytotoxicity ofIFNτ which makes it extremely valuable as an in vivo therapeutic agent.This lack of cytotoxicity sets IFNτ apart from most other knownantiviral agents and all other known interferons.

Formulations comprising the IFNτ compounds of the present invention canbe used to inhibit viral replication.

The human IFNτ of the present invention can be employed in methods foraffecting the immune relationship between fetus and mother, for example,in preventing transmission of maternal viruses (e.g., HIV) to thedeveloping fetus. The human interferon-τ is particularly useful fortreatment of humans, since potential antigenic responses are less likelyusing a homologous protein.

C. Anticellular Proliferation Properties

IFNτ exhibits potent anticellular proliferation activity. IFNτ can alsobe used to inhibit cellular growth without the negative side effectsassociated with other interferons which are currently known.Formulations comprising the IFNτ compounds of the subject invention canbe used to inhibit, prevent, or slow tumor growth.

The development of certain tumors is mediated by estrogen. Experimentsperformed in support of the present invention indicate that IFNτ cansuppress estrogen receptor numbers. Therefore, IFNτ can be used in thetreatment or prevention of estrogen-dependent tumors.

D. Interfering with the Binding of Interferons to Receptors

IFNτ appears to interact with the Type I IFN receptor via severalepitopes on the molecule, and these regions either separately or incombination may affect distinct functions of IFNτ differently.

The polypeptides of the present invention are useful for the selectiveinhibition of binding of interferons to the interferon receptor.Specifically, as described herein, certain of the disclosed peptidesselectively inhibit the antiviral activity of IFNτ while others inhibitthe antiproliferative activity. Combinations of these peptides could beused to inhibit both activities. Advantageously, despite binding to theinterferon receptor and blocking IFNτ activity, these peptides do not,themselves, elicit the antiviral or antiproliferative activity.

Therefore, such polypeptides can be used as immunoregulatory moleculeswhen it is desired to prevent immune responses triggered by interferonmolecules. These peptides could be used as immunosuppressants toprevent, for example, interferon-mediated immune responses to tissuetransplants. Other types of interferon mediated responses may also beblocked, such as the cytotoxic effects of alpha interferon.

E. Pharmaceutical Compositions

IFNτ proteins can be formulated according to known methods for preparingpharmaceutically useful compositions. Formulations comprisinginterferons or interferon-like compounds have been previously described(for example, Martin, 1976). In general, the compositions of the subjectinvention will be formulated such that an effective amount of the IFNτis combined with a suitable carrier in order to facilitate effectiveadministration of the composition.

The compositions used in these therapies may also be in a variety offorms. These include, for example, solid, semi-solid, and liquid dosageforms, such as tablets, pills, powders, liquid solutions or suspensions,liposomes, suppositories, injectable, and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically acceptable carriers and adjuvants whichare known to those of skill in the art. Preferably, the compositions ofthe invention are in the form of a unit dose and will usually beadministered to the patient one or more times a day.

IFNτ, or related polypeptides, may be administered to a patient in anypharmaceutically acceptable dosage form, including oral intake,inhalation, intranasal spray, intraperitoneal, intravenous,intramuscular, intralesional, or subcutaneous injection. Specifically,compositions and methods used for other interferon compounds can be usedfor the delivery of these compounds.

One primary advantage of the compounds of the subject invention,however, is the extremely low cytotoxicity of the IFNτ proteins. Becauseof this low cytotoxicity, it is possible to administer the IFNτ inconcentrations which are greater than those which can generally beutilized for other interferon (e.g., IFNα) compounds. Thus, IFNτ can beadministered at rates from about 5×10⁴ to 20×10⁶ units/day to about500×10⁶ units/day or more. In a preferred embodiment, the dosage isabout 20×10⁶ units/day. High doses are preferred for systemicadministration. It should, of course, be understood that thecompositions and methods of this invention may be used in combinationwith other therapies.

Once improvement of a patient's condition has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, may be reduced, as a function ofthe symptoms, to a level at which the improved condition is retained.When the symptoms have been alleviated to the desired level, treatmentshould cease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

The compositions of the subject invention can be administered throughstandard procedures to treat a variety of cancers and viral diseasesincluding those for which other interferons have previously shownactivity. See, for example, Finter, et al. (1991); Dianzani, et al.(1992); Francis, et al. (1992) and U.S. Pat. Nos. 4,885,166 and4,975,276. However, as discussed above, the compositions of the subjectinvention have unique features and advantages, including their abilityto treat these conditions without toxicity.

F. Treatment of Skin Disorders

Disorders of the skin can be treated intralesionally using IFNτ, whereinformulation and dose will depend on the method of administration and onthe size and severity of the lesion to be treated. Preferred methodsinclude intradermal and subcutaneous injection. Multiple injections intolarge lesions may be possible, and several lesions on the skin of asingle patient may be treated at one time. The schedule foradministration can be determined by a person skilled in the art.Formulations designed for sustained release can reduce the frequency ofadministration.

G. Systemic Treatment

Systemic treatment is essentially equivalent for all applications.Multiple intravenous, subcutaneous and/or intramuscular doses arepossible, and in the case of implantable methods for treatment,formulations designed for sustained release are particularly useful.Patients may also be treated using implantable subcutaneous portals,reservoirs, or pumps.

H. Regional Treatment

Regional treatment with the IFNτ polypeptides of the present inventionis useful for treatment of cancers in specific organs. Treatment can beaccomplished by intraarterial infusion. A catheter can be surgically orangiographically implanted to direct treatment to the affected organ. Asubcutaneous portal, connected to the catheter, can be used for chronictreatment, or an implantable, refillable pump may also be employed.

The following examples illustrate, but in no way are intended to limitthe present invention.

Materials and Methods

Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, TaqDNA polymerase, and calf intestinal phosphatase were purchased from NewEngland Biolabs (Beverly, Mass.) or Promega Biotech (Madison, Wis.):these reagents were used according to the manufacturer's instruction.For sequencing reactions, a "SEQUENASE DNA II" sequencing kit was used(United States Biochemical Corporation, Cleveland, Ohio). Immunoblottingand other reagents were from Sigma Chemical Co. (St. Louis, Mo.) orFisher Scientific (Needham, Mass.). Nitrocellulose filters are obtainedfrom Schleicher and Schuell (Keene, N.H.).

Synthetic oligonucleotide linkers and primers are prepared usingcommercially available automated oligonucleotide synthesizers (e.g., anABI model 380B-02 DNA synthesizer (Applied Biosystems, Foster City,Calif.)). Alternatively, custom designed synthetic oligonucleotides maybe purchased, for example, from Synthetic Genetics (San Diego, Calif.).cDNA synthesis kit and random priming labeling kits are obtained fromBoehringer-Mannheim Biochemical (BMB, Indianapolis, Ind.).

Oligonucleotide sequences encoding polypeptides can be eithersynthesized directly by standard methods of oligonucleotide synthesis,or, in the case of large coding sequences, synthesized by a series ofcloning steps involving a tandem array of multiple oligonucleotidefragments corresponding to the coding sequence (Crea; Yoshio et al.;Eaton et al.). Oligonucleotide coding sequences can be expressed bystandard recombinant procedures (Maniatis et al.; Ausubel et al.).

Alternatively, peptides can be synthesized directly by standard in vitrotechniques (Applied Biosystems, Foster City Calif.).

Common manipulations involved in polyclonal and monoclonal antibodywork, including antibody purification from sera, are performed bystandard procedures (Harlow et al.). Pierce (Rockford, Ill.) is a sourceof many antibody reagents.

Recombinant human IFNα (rHuIFNα) and rBoIFNγ was obtained from GenentechInc. (South San Francisco, Calif.). The reference preparation ofrecombinant human IFNα (rHuIFNα) was obtained from the NationalInstitutes of Health: rHuIFNα is commercially available from LeeBiomolecular (San Diego, Calif.).

All tissue culture media, sera and IFNs used in this study were negativefor endotoxin, as determined by assay with Limulus amebocyte lysate(Associates of Cape Cod, Woods Hole, Mass.) at a sensitivity level of0.07 ng/ml.

General ELISA Protocol for Detection of Antibodies

Polystyrene 96 well plates Immulon II (PGC) were coated with 5 μg/mL(100 μL per well) antigen in 0.1M carb/bicarbonate buffer, pH 9.5.Plates were sealed with parafilm and stored at 4° C. overnight.

Plates were aspirated and blocked with 300 uL 10% NGS and incubated at37° C. for 1 hr.

Plates were washed 5 times with PBS 0.5% "TWEEN-20".

Antisera were diluted in 0.1M PBS, pH 7.2. The desired dilution(s) ofantisera (0.1 mL) were added to each well and the plate incubated 1hours at 37° C. The plates was then washed 5 times with PBS 0.5%"TWEEN-20".

Horseradish peroxidase (HRP) conjugated goat antihuman antiserum(Cappel) was diluted 1/5,000 in PBS. 0.1 mL of this solution was addedto each well. The plate was incubated 30 min at 37° C., then washed 5times with PBS.

Sigma ABTS (substrate) was prepared just prior to addition to the plate.

The reagent consists of 50 mL 0.05M citric acid, pH 4.2, 0.078 mL 30%hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of the substrate wasadded to each well, then incubated for 30 min at room temperature. Thereaction was stopped with the addition of 0.050 mL 5% SDS (w/v). Therelative absorbance is determined at 410 nm.

EXAMPLE 1 Reproductive Functions of IFNτ

The effect of interferon-τ on the lifespan of the corpus lutem wasexamined.

IFNτ was infused into uterine lumen of ewes at the concentrations givenin Table 1. Recombinant human IFNα (rHuIFNα) was infused at similarconcentrations. In addition, control animals, which received controlproteins, were also used. The life span of the corpus luteum wasassessed by examination of interestrous intervals, maintenance ofprogesterone secretion, and inhibition of prostaglandin secretion(Davis, et al., 1992).

                  TABLE 1    ______________________________________    Effect of Interferons on Reproductive Physiology                          Interestrous Interval (days)    Interferon Treatment  (Means)    ______________________________________    Control    --         17.3    rHuIFNα               100 μg/day                          16.0               200 μg/day                          16.0               2000 μg/day                          19.0    OvIFNτ 100 μg/day                          27.2    ______________________________________

Comparison of the interestrous intervals for the control animals and foranimals receiving OvIFNτ demonstrate a considerable lengthening of theinterval, when IFNτ is administered at 100 μg/day. On the other hand,comparison of the interestrous interval for the control animal and foranimals receiving recombinant human IFNα, demonstrated that rHuIFNα hadno meaningful effect.

These results demonstrate that interferon-τ has the capability ofsignificantly influencing the biochemical events of the reproductivecycle.

EXAMPLE 2 Antiviral Properties of Interferon-τ at Various Stages of theReproductive Cycle

Conceptus cultures were established using conceptus obtained from sheepat days 12 through 16 of the estrous cycle. Antiviral activity ofsupernatant from each conceptus culture was assessed using a cytopathiceffect assay (Familetti, et al., 1981). Briefly, dilutions of IFNτ orother IFNs were incubated with Madin-Darby bovine kidney (MDBK) cellsfor 16-18 hours at 37° C. Following incubation, inhibition of viralreplication was determined in a cytopathic effect assay using vesicularstomatitis virus (VSV) as the challenge virus.

One antiviral unit caused a 50% reduction in destruction of themonolayer, relative to untreated MDBK cells infected with VSV (controlplates). Specific activities were further evaluated using normal ovinefibroblasts (Shnf) in a plaque inhibition assay (Langford, et al.,1981). A minimum of three samples were examined at each time point, andeach sample was assayed in triplicate. The results presented in Table 2are expressed as mean units/ml.

                  TABLE 2    ______________________________________    IFNτ Antiviral Activity of Conceptus Cultures    and Allantoic and Amniotic Fluids               Day      Samples Units/ml    ______________________________________    Conceptus Cultures                 10         9       <3                 12         5       34                 13         6       4.5 × 10.sup.3                 14         3       7.7 × 10.sup.3                 16         12      2.0 × 10.sup.6    Allantoic Fluid                 60         3       1.4 × 10.sup.3                 100        4       11                 140        3       <3    Amniotic Fluid                 60         3       22                 100        4       <3    ______________________________________

Culture supernatants had increasing antiviral activity associated withadvancing development of the conceptus (Table 2).

EXAMPLE 3 Expression of IFNτ in Bacteria

The amino acid coding sequence for OvIFNτ (Imakawa, et al., 1987) wasused to generate a corresponding DNA coding sequence with codon usageoptimized for expression in E. coli. Linker sequences were added to the5' and 3' ends to facilitate cloning in bacterial expression vectors.The nucleotide sequence was designed to include 19 unique restrictionenzyme sites spaced evenly throughout the coding sequence (FIGS. 1A and1B).

The nucleotide sequence was divided into eleven oligonucleotidefragments ranging in sizes of 33 to 75 bases. Each of the elevenoligonucleotides were synthesized on a 380-B 2-column DNA synthesizer(Applied Biosystems) and cloned single- or double-stranded into one ofthe following vectors: "pBLUESCRIPT⁺ (KS)" (Stratagene, LaJolla,Calif.), pTZ18R (Pharmacia, Piscataway, N.J.), or pTZ19R (Pharmacia,Piscataway, N.J.) cloning vectors.

The vectors were transformed into E. coli K. strain "XL1-BLUE" (recA1endA1 gyrA96 thi hsdR17 (r_(k) ⁻, m_(k) +) supE44 relA1 λ-(lac), {F',proAB, lac^(q) ZΔM15, Tn10(tet^(R) }) which is commercially availablefrom Stratagene (La Jolla, Calif.). Transformed cells were grown in Lbroth supplemented with ampicillin (50 μg/ml). Oligonucleotide cloningand fusion was performed using standard recombinant DNA techniques.

Cloning vectors were cut with the appropriate restriction enzymes toinsert the synthetic oligonucleotides. The vectors were treated withcalf intestine alkaline phosphatase (CIP) to remove terminal phosphategroups. Oligonucleotides were phosphorylated and cloned, as eithersingle- or double-stranded molecules, into the appropriate vector usingT4 DNA ligase. When single-strands were introduced into cloning vectors,the second strand was completed by the bacterial host followingtransfection.

For double-stranded cloning, oligonucleotides were first annealed withtheir synthetic complementary strand then ligated into the cloningvector. E. coli K12 strains SB221 or NM522 were then transformed withthe ligation. E. coli strain GM119 was used for cloning when themethylation-sensitive StuI and ClaI restriction sites were involved.Restriction analyses were performed on isolated DNA at each stage of thecloning procedure.

Cloned oligonucleotides were fused into a single polynucleotide usingthe restriction digestions and ligations outlined in FIG. 2.oligonucleotide-containing-DNA fragments were typically isolated afterelectrophoretic size fractionation on low-melting point agarose gels(Maniatis, et al.; Sambrook, et al.; Ausubel, et al.). The resultingIFNτ polynucleotide coding sequence spans position 16 through 531: acoding sequence of 172 amino acids.

The nucleotide sequence of the final polynucleotide was confirmed by DNAsequencing using the dideoxy chain termination method.

The full length StuI/SstI fragment (540 bp; FIG. 2) was cloned into amodified pIN III omp-A expression vector and transformed into acompetent SB221 strain of E. coli. For expression of the IFNτ protein,cells carrying the expression vector were grown in L-broth containingampicillin to an OD (550 nm) of 0.1-1, induced with IPTG for 3 hours andharvested by centrifugation. Soluble recombinant IFNτ was liberated fromthe cells by sonication or osmotic fractionation.

EXAMPLE 4 Expression of IFNτ in Yeast

The synthetic IFNτ gene, synthesized in Example 3, was flanked at the 5'end by an StuI restriction site and at the 3' end by a SacI restrictionsite.

A. Isolation of the Synthetic IFNτ Gene

Two oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) were used toattach linkers to the synthetic IFNτ gene using polymerase chainreaction. The linker at the 5' end allowed the placement of thesynthetic IFNτ gene in correct reading with the ubiquitin codingsequence present in the yeast cloning vector pBS24Ub (Chiron Corp.,Emeryville, Calif.). The linker also constructed a ubiquitin-IFNτjunction region that allowed in vivo cleavage of the ubiquitin sequencesfrom the IFNτ sequences. The 5' oligonucleotide also encoded a SacIIrestriction endonuclease cleavage site. The 3' oligonucleotide containeda StuI cleavage site.

The vector carrying the synthetic IFNτ gene (Example 3) was isolatedfrom E. coli strain "XLI-BLUE" by the alkaline lysis method. Isolatedvector was diluted 500-fold in 10 mM Tris, pH 8.0/1 mM EDTA/10 mM NaCl.The PCR reaction was performed in a 100 μl volume using Taq DNApolymerase and primers SEQ ID NO:13/SEQ ID NO:14. The amplifiedfragments were digested with StuI and SacII. These digested fragmentswere ligated into the SacII and SmaI sites of "pBLUESCRIPT+(KS)."

The resulting plasmid was named pBSY-IFNτ. The DNA sequence was verifiedusing double stranded DNA as the template.

B. Construction of the Expression Plasmid

Plasmid pBSY-IFNτ was digested with SacII and EcoRV and the fragmentcontaining the synthetic IFNτ gene was isolated. The yeast expressionvector pBS24Ub (Sabin, et al.; Ecker, et al.) was digested with SalI.Blunt ends were generated using T4 DNA polymerase. The vector DNA wasextracted with phenol and ethanol precipitated (Sambrook, et al., 1989).The recovered linearized plasmid was digested with SacII, purified byagarose gel electrophoresis, and ligated to the SacII-EcoRV fragmentisolated from pBSY-IFNτ. The resulting recombinant plasmid wasdesignated pBS24Ub-IFNτ.

The recombinant plasmid pBS24Ub-IFNτ was transformed into E. coli.Recombinant clones containing the IFNτ insert were isolated andidentified by restriction enzyme analysis. Plasmid DNA from clonescontaining IFNτ coding sequences was used for transformation of S.cerevisiae (Rothstein, 1986). Transformation mixtures were plated onuracil omission medium and incubated for 3-5 days at 30° C. Colonieswere then streaked and maintained on uracil and leucine omission medium(Rothstein, 1986).

C. Expression Experiments

For small-scale expression, a single colony of S. cerevisiae AB116containing pBS24Ub-IFNτ was picked from a leucine and uracil omissionplate and grown at 30° C. in YEP medium (1% yeast extract, 2% peptone)containing 1% glucose for inducing conditions or 8% glucose fornoninducing conditions. Cell lysates were recovered and subjected toSDS-PAGE in 15% acrylamide, 0.4% bisacrylamide (Sambrook, et al., 1989).The fractionated proteins were visualized by Coomassie blue staining.

Recombinant IFNτ was visualized specifically by immunoblotting withmonoclonal antibody or polyclonal antiserum against ovine IFNτ uponelectrotransfer of the fractionated cell extract to "NYTRAN" paper(Rothstein, 1986).

For large-scale expression, pBS24-IFNτ was grown for 24 hours at 30° C.in 5×uracil and leucine omission medium containing 8% glucose. Thisculture was then diluted 20-fold in YEP medium containing 1% glucose andfurther incubated for another 24-36 hours.

Cells were harvested by centrifugation, washed in 50 mM Tris, pH 7.6,/1mM EDTA and resuspended in wash buffer containing 1 mM PMSF. The cellswere lysed using a Bead-beater apparatus (Biospec Products,Bartlesville, Okla.). The lysate was spun at 43,000×g for 20 minutes.The supernatant fraction was recovered and subjected to the purificationprotocol described below.

D. Purification of roIFNτ from Yeast Cell Lysate

The supernatant was loaded on a 1×10 cm DEAE column and washed with 10mM Tris, pH 8.0. Retained proteins were eluted with a 300 ml, 0 to 0.5MNaCl gradient in 10 mM Tris, pH 8.0. Three-milliliter fractions werecollected. Ten-microliter samples of fractions 17-26 containing therecombinant (roIFNτ) were electrophoretically separated on 15%SDS-polyacrylamide gels. The gels were stained with Coomassie blue.

Fractions 18, 19, and 20 contained largest amount of roIFNτ. Thesefractions were loaded individually on a 1.5×90 cm Sephadex S-200 columnand proteins were resolved in two peaks. Aliquots of each protein peak(25 μl) were electrophoretically separated on 15% SDS-polyacrylamidegels and the proteins visualized with Coomassie staining.

Purified roIFNτ-containing fractions were combined and the amount ofroIFNτ quantified by radioimmunoassay (Vallet, et al., 1988). Totalprotein concentration was determined by using the Lowry protein assay(Lowry, et al., 1951).

Microsequencing of purified roIFNτ demonstrated identity with nativeIFNτ through the first 15 amino acids, confirming that theubiquitin/roIFNτ fusion protein was correctly processed in vivo.

Purified roIFNτ exhibited 2 to 3×10⁸ units of antiviral activity permilligram of protein (n=3 replicate plates) which is similar to theantiviral activity of IFNτ purified from conceptus-conditioned culturemedium (2×10⁸ U/mg).

EXAMPLE 5 Southern Blot Analysis of Human High Molecular Weight DNA

Human venous blood samples from healthy donors were collected inheparinized tubes and peripheral blood lymphocytes were isolated bydensity-gradient centrifugation using a Ficoll-Isopaque gradient (1.077g/ml) (Sigma Chemical Co.). High molecular weight (HMW) DNA was isolatedfrom these cells (Sambrook, et al., 1989).

Two 10 μg samples of HMW DNA were digested with the restrictionendonucleases HindIII or PstI (Promega) for 2 hours at 37° C., and theDNA fragments electrophoretically separated in a 0.8% agarose gel(Bio-Rad, Richmond, Calif.) at 75 volts for 8 hours. The DNA fragmentswere transferred onto a nylon membrane (IBI-InternationalBiotechnologies, Inc., New Haven, Conn.). The membrane was baked at 80°C. for 2 hours and incubated at 42° C. for 4 hours in the followingprehybridization solution: 5×SSC (1×SSC is 0.15M NaCl and 0.15M sodiumcitrate), 50% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfatdry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 0.5 mg/ml singlestranded herring sperm DNA (Promega).

The filter was then incubated in a hybridization solution (5×SSC, 20%vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk, 20mM Tris-HCl (pH 7.5), 4 mM EDTA, and 2×10⁸ cpm/ml ³² P-labelled OvIFNτcDNA (Imakawa, et al., 1987)) for 18 hours at 42° C. The filter waswashed at 42° C. for 15 minutes with 2×SSC and 0.1% (wt/vol) SDS andexposed to X-ray film (XAR, Eastman Kodak, Rochester, N.Y.) at -80° C.for 48 hours in the presence of an intensifying screen.

Autoradiography detected a hybridization signal at approximately 3.4 kbin DNA digested with PstI and a slightly smaller (≈3.0 kb) fragment inthe HindIII digested DNA. These results indicate the presence of humanDNA sequences complementary to the OvIFNτ cDNA probe.

EXAMPLE 6 Isolation of Partial Sequence of Human IFNτ cDNA by PCR

Two synthetic oligonucleotides (each 25-mer), corresponding to thenucleotides in the DNA sequence from 231 to 255 (contained in SEQ IDNO:13) and 566 to 590 (contained in SEQ ID NO:14) of OvIFNτ cDNA(numbering relative to the cap site, Imakawa, et al., 1987) weresynthesized. These primers contained, respectively, cleavage sites forthe restriction endonucleases PstI and EcoRI. SEQ ID NO:13 was modifiedto contain the EcoRI site, which begins at position 569.

DNA was isolated from approximately 1×10⁵ plaque forming units (pfu) ofthe following two cDNA libraries: human term placenta (Clontech, Inc.,Palo Alto, Calif.) and human term cytotrophoblast (Dr. J. F. Strauss,University of Pennsylvania, Philadelphia, Pa.). The DNA was employed inpolymerase chain reaction (PCR) amplifications (Mullis; Mullis, et al.;Perkin Elmer Cetus Corp. Norwalk, Conn.). Amplification reactions werecarried out for 30 cycles (45° C., 1 m; 72° C., 2 m; 94° C., 1 m)(thermal cycler and reagents, Perkin Elmer Cetus) using primers SEQ IDNO:13/SEQ ID NO:14.

Amplification products were electrophoretically separated (100 volts ina 1.5% agarose gel (Bio-Rad)) and transferred onto a nylon membrane(IBI). The membrane was baked at 80° C. for 2 hours and prehybridizedand hybridized with ³² P-labelled OvIFNτ cDNA as described above. Themembrane was washed in 5×SSC/0.1% (wt/vol) SDS for 5 minutes at 42° C.and in 2×SSC/0.1% (wt/vol) SDS for 2 minutes at 42° C. It was thenexposed at -80° C. to "XAR" (Eastman Kodak) X-ray film for 24 hours inthe presence of an intensifying screen. An amplification product thathybridized with the labelled probe DNA was detected.

PCR was performed again as directed above. Amplified products weredigested with the restriction endonucleases EcoRI and PstI (Promega) for90 minutes at 37° C. The resulting DNA fragments wereelectrophoretically separated as described above and the band containingthe IFNτ amplification product was excised from the gel. DNA fragmentswere recovered by electroelution, subcloned into EcoRI/PstIdigested-dephosphorylated plasmid pUC19 and transformed into E. colistrain JM101 (Promega) by calcium chloride method (Sambrook, et al.,1989). The plasmids were isolated and the inserted amplification productsequenced using the dideoxy termination method (Sanger, et al., 1977;"SEQUENASE" reactions, United States Biochemical, Cleveland, Ohio).Nucleotide sequences were determined, and comparison of these as well asthe deduced amino acid sequences to other IFN sequences were performedusing "DNA STAR SOFTWARE" (Madison, Wis.).

Comparison of the sequences of these clones revealed the following fourdifferent clones: from the human placental library, HuIFNτ6 (299 bp),HuIFNτ7 (288 bp) and HuIFNτ4 (307 bp), which exhibit 95% identity intheir nucleotide sequences; from the cytotrophoblast library clone CTB35 (HuIFNτ5; 294 basepairs), which shares 95% and 98% identity withHuIFNτ6 and HuIFNτ4, respectively.

EXAMPLE 7

Isolation of Full-Length Human IFNτ Genes

Ten micrograms PBMC HMW DNA was digested with restriction endonucleaseEcoRI and subjected to electrophoretic analysis in a 0.8% agarose gel. Aseries of samples containing ranges of DNA fragments sized 1.5 to 10 kb(e.g., 1.5 to 2.5 kb, 2.5 kb to 3 kb) were excised from the gel. TheDNAs were electroeluted and purified. Each DNA sample was amplified asdescribed above using the OvIFNτ primers. The DNA molecules of anysample that yielded a positive PCR signal were cloned into λgt11 (thesubgenomic λgt11 library).

A. PCR Identification of Clones Containing Sequences Complementary toOvIFNτ

The λgt11 phage were then plated for plaques and plaque-lifthybridization performed using the ³² P-labelled OvIFNτ cDNA probe.Approximately 20 clones were identified that hybridized to the probe.

Plaques that hybridized to the probe were further analyzed by PCR usingthe OvIFNτ primers described above. Six plaques which generated positivePCR signals were purified. The phage DNA from these clones was isolatedand digested with EcoRI restriction endonuclease. The DNA inserts weresubcloned into pUC19 vectors and their nucleotide sequences determinedby dideoxy nucleotide sequencings.

B. Hybridization Identification of Clones Containing SequencesComplementary to PCR-Positive Phage

Recombinant phage from the λgt11 subgenomic library were propagated inE. coli Y1080 and plated with E. coli Y1090 at a density of about 20,000plaques/150 mm plate. The plates were overlaid with duplicatenitrocellulose filters, which were hybridized with a ³² P-labelled probefrom one of the six human IFNτ cDNA clones isolated above.

Clones giving positive hybridization signals were further screened andpurified. The phage DNAs from hybridization-positive clones wereisolated, digested with EcoRI, subcloned into pUC19 vector andsequenced. The sequence information was then analyzed.

1. HuIFNτ1

Three clones yielded over-lapping sequence information for over 800bases relative to the mRNA cap site (clones were sequenced in bothorientations). The combined nucleic acid sequence information ispresented as SEQ ID NO:11 and the predicted protein coding sequence ispresented as SEQ ID NO:12. Comparison of the predicted mature proteinsequence (SEQ ID NO:12) of this gene to the predicted protein sequenceof OvIFNτ is shown in FIG. 3.

2. HuIFNτ2, HuIFNτ3

Two additional clones giving positive hybridization signals (HuIFNτ2 andHuIFNτ3) were also screened, purified, and phage DNAs subcloned andsequenced as above. The sequences of these two clones are presented inFIGS. 19A and 19B. As can be appreciated in FIGS. 19A and 19B, thenucleotide sequence of both clones (HuIFNτ2 and HuIFNτ3) is homologousto that of HuIFNτ1 and OvIFNτ.

HuIFNτ2 (SEQ ID NO:29), may be a pseudo-gene, as it appears to contain astop codon at position 115-117. The sequence, SEQ ID NO:29, is presentedwithout the leader sequence. The leader sequence is shown in FIG. 20A.As can be seen from the HuIFNτ2 sequence presented in FIG. 20A, thefirst amino acid present in mature HuIFNτ1 (a CYS residue) is notpresent in the HuIFNτ2 sequence. Accordingly, the predicted amino acidsequence presented as SEQ ID NO:29 corresponds to a mature IFNτ proteinwith the exceptions of the first CYS residue and the internal stopcodon.

The internal stop codon in the nucleic acid coding sequence can bemodified by standard methods to replace the stop codon with an aminoacid codon, for example, encoding GLN. The amino acid GLN is present atthis position in the other isolates of human IFNτ (HuIFNτ). Standardrecombinant manipulations also allow introduction of the initial CYSresidue if so desired.

HuIFNτ3 (SEQ ID NO:31), appears to encode a human IFNτ protein. Thetranslated amino acid sequence of the entire protein, including theleader sequence, is presented as SEQ ID NO:32. The translated amino acidsequence of the mature protein is presented as SEQ ID NO:34.

EXAMPLE 8 Analysis of the Presence of HuIFNτ mRNA by RT-PCR

Human placental cDNA libraries and an ovine cDNA library, constructedfrom day 15-16 conceptuses, were analyzed by hybridization to the OvIFNτcDNA probe, described above. cDNAs were size-fractionated on agarosegels and transferred to filters (Maniatis, et al.; Sambrook, et al.).Southern blot analysis with OvIFNτ probe showed that theautoradiographic signals from human cDNA libraries were approximately1/100 of the signal obtained using the OvIFNτ cDNA library.

The presence of HuIFNτ mRNA in human term placenta and amniocytes (26weeks, 2 million cells) was analyzed by using reverse transcriptase-PCR(RT-PCR) method (Clontech Laboratories, Palo Alto, Calif.).

Total cellular RNA (tcRNA) isolated from human placenta, amniocytes andovine conceptuses were reverse transcribed using the primer SEQ IDNO:14. The primer SEQ ID NO:13 was then added to the reaction andpolymerase chain reaction carried out for 40 cycles. The PCR productswere size fractionated on agarose gels and transferred to filters. TheDNA on the filters was hybridized with ³² P-labelled OvIFNτ and HuIFNτcDNAs. The results of these analyses demonstrate the presence of humanIFNτ mRNA in the feto-placental annex. The aminocytes also expressed themessages corresponding to OvIFNτ primers and human probe.

In addition, a RT-PCR analysis for the presence of HuIFNτ was applied tothe tcRNA isolated from human adult lymphocytes. A densitometricanalysis revealed that IFNτ mRNA exists in lymphocytes.

EXAMPLE 9 In Situ Hybridization

A. Tissue

Slides of semiserial 5-μ paraffin embedded sections from four healthy,different term and first trimester human placentas were examined.

B. cRNA Probe Preparation

From the cDNA clone isolated from OvIFNτ amplified library a fragmentcorresponding to the OvIFNτ cDNA bases #77-736 (base #1 is cap site;open reading frame of OvIFNτ cDNA is base #81-665; FIG. 7) was subclonedinto the transcription vector, pBS (New England Biolabs). Several pBSclones were isolated, subcloned, and their nucleotides sequenced. Fromthis clone a 3' fragment (bases #425-736) was excised using therestriction endonucleases NlaIV and EcoRI and subcloned into thetranscription vector pBS. This vector was designated pBS/OvIFNτ.

After linearization of the pBS/OvIFNτ plasmid, an antisense cRNA probewas synthesized by in vitro transcription (Sambrook, et al., 1989) usingT₇ RNA polymerase (Stratagene). A trace amount of ³ H-CTP (NEN-DuPont,Cambridge, Mass.) was used in the transcription reaction. dUTP labeledwith digoxigenin (Boehringer-Mannheim, Indianapolis, Ind.) wasincorporated into the cRNA and yield was estimated through TCAprecipitation and scintillation counting.

C. Hybridization

In situ hybridization was performed using the anti-sense RNA probe, asdescribed by Lawrence, et al. (1985) with the following modifications.Deparaffinized and hydrated sections were prehybridized for 10 minutesat room temperature in phosphate buffered saline (PBS) containing 5 mMMgCl₂. Nucleic acids in the sections were denatured for 10 minutes at65° C. in 50% formamide/2×SSC. Sections were incubated overnight at 37°C. with a hybridization cocktail (30 μl/slide) containing 0.3 μg/mldigoxigenin-labelled cRNA probe and then washed for 30 minutes each at37° C in 50 formamide/1×SSC. Final washes were performed for 30 minuteseach at room temperature in 1×SSC and 0.1×SSC. The sections were blockedfor 30 minutes with 0.5% Triton X-100 (Sigma) and 0.5% non-fat dry milk.

Hybridization signal was detected using purified sheep antidioxigeninFab fragments conjugated to alkaline phosphatase (Boehringer-Mannheim).After unbound antibody was removed, nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Promega) andlevamisole (Bector Laboratories, Burlingame, Calif.) were added forsignal detection via colorimetric substrate generation. The tissues werecounterstained in methyl green (Sigma), dehydrated, and mounted.

As a control, some tissue sections were pretreated with 100 μg/ml ofpancreatic RNaseA (Sigma) for 30 minutes at 37° C. The RNase wasinactivated on the slide with 400 units of RNase inhibitor (Promega).The slides were then washed twice in 250 ml of PBS/5 mM MgCl₂. In othercontrol experiments, tRNA (Sigma) was substituted for the digoxigeninprobes.

Specific hybridization was observed in all term and first trimesterplacental tissues in three separate experiments with various OvIFNτ cRNAprobe concentrations and blocking reagents.

First trimester placental villi composed of an outer layer ofsyncytiotrophoblast, an underlying layer of cytotrophoblast, and acentral stromal region with various types of mesenchymal cells,displayed the highest transcript level of IFNτ in the cytotrophoblastcells. Less intense but detectable levels were present in both thesyncytiotrophoblast and stromal cells. A similar pattern of transcriptexpression was demonstrated in the placental villi of term tissue butthe level of signal detection was low. First trimester extravilloustrophoblast displayed the highest amount of message and stained positivewhen present in the maternal blood spaces.

EXAMPLE 10 Antiviral Activity of IFNτ

The relative specific activity of OvIFNτ, purified to homogeneity, wasevaluated in antiviral assays. The antiviral assays were performedessentially as described above in Example 2. Specific activities areexpressed in antiviral units/mg protein obtained from antiviral assaysusing either Madin-Darby bovine kidney (MDBK) cells or sheep normalfibroblasts (Shnf). All samples were assayed simultaneously to eliminateinterassay variability. The results, presented in Table 3, are the meansof four determinations where the standard deviation was less than 10% ofthe mean.

                  TABLE 3    ______________________________________    Antiviral Activity of IFNτ and Known IFNs                  Specific Activities                  MDBK   Shnf    ______________________________________    OvIFNτ        2 × 10.sup.8                             3 × 10.sup.8    rBoIFNα     6 × 10.sup.7                             1 × 10.sup.7    rBoIFN.sub.γ                    4.5 × 10.sup.6                             3 × 10.sup.6    NIH rHuIFNα                    2.2 × 10.sup.8                             2.2 × 10.sup.8    rHuIFNα   2.9 × 10.sup.5                             4.3 × 10.sup.5    ______________________________________

IFNτ had a higher specific activity than either rBoIFNα or rBoIFNγ(Table 3). The NIH standard preparation of rHuIFNα had a similarspecific activity, while a commercial preparation of rHuIFNα exhibitedlow specific antiviral activity. Comparable relative antiviral activitywas demonstrated using either bovine or ovine cells.

EXAMPLE 11 Anti-Retroviral Activity and Cytotoxic Effects of IFNτ

Highly purified OvIFNτ was tested for anti-retroviral and cytotoxiceffects on feline peripheral blood lymphocytes exposed to the felineimmunodeficiency retrovirus. This lentivirus produces a chronicAIDS-like syndrome in cats and is a model for human AIDS (Pederson, etal., 1987). Replication of the virus in peripheral blood lymphocytes ismonitored by reverse transcriptase activity in culture supernatants overtime. The data from these assays are presented in Table

                  TABLE 4    ______________________________________    Effect of OvIFNτ on FIV Replication    ______________________________________    IFNτ Concentration                 RT Activity (cpm/ml)    (ng/ml)      Harvest Days    Experiment 1 Day 2   Day 5   Day 8 Day 12                                             Day 15    ______________________________________    0.00         93,908  363,042 289,874                                       171,185                                             125,400    0.62         77,243  179,842 172,100                                       218,281                                             73,039    1.25         94,587  101,873 122,216                                       71,916                                             50,038    2.50         63,676  72,320  140,783                                       75,001                                             36,105    5.00         69,348  82,928  90,737                                       49,546                                             36,299    ______________________________________               Harvest Days    Experiment 2 Day 2   Day 5   Day 8 Day 13                                             Day 17    ______________________________________    0.0          210,569 305,048 279,556                                       500,634                                             611,542    2.5          121,082 106,815 108,882                                       201,676                                             195,356    5.0          223,975 185,579 108,114                                       175,196                                             173,881    10.0         167,425 113,631 125,131                                       131,649                                             129,364    20.0         204,879 80,399  59,458                                       78,277                                             72,179    40.0         133,768 54,905  31,606                                       72,580                                             53,493    ______________________________________

Addition of OvIFNτ produced a rapid, dose-dependent decrease in reversetranscriptase (RT) activity (Table 4). While concentrations as low as0.62 ng/ml of IFNτ inhibited viral replication, much higherconcentrations (40 ng/ml) having greater effects on RT-activity werewithout toxic effects on the cells. The results suggest that replicationof the feline immunodeficiency virus was reduced significantly comparedto control values when cells were cultured in the presence of OvIFNτ.

IFNτ appeared to exert no cytotoxic effect on the cells hosting theretrovirus. This was true even when IFNτ was present at 40 ng per ml ofculture medium.

EXAMPLE 12 Effects of IFNτ on HIV Infected Human Peripheral Lymphocytes

IFNτ was also tested for activity against HIV infection in human cells.Human peripheral blood lymphocytes, which had been infected with HIV(Crowe, et al.), were treated with varying concentrations of OvIFNτ.Replication of HIV in peripheral blood lymphocytes was monitored byreverse transcriptase activity in culture supernatants over time.Reverse transcriptase activity was measured essentially by the method ofHoffman, et al. The data from these assays are presented in Table 5.

                  TABLE 5    ______________________________________    Effect of OvIFNτ on HIV Replication in    Human Peripheral Lymphocytes    IFNτ RT Activity    Concentration             Day 6           Day 10    (ng/ml)  cpm/ml   % Reduction                                 cpm/ml % Reduction    ______________________________________    0        4,214    --         25,994 --    10       2,046    51         9,883  62    50       1,794    57         4,962  81    100      1,770    58         3,012  88    500      1,686    60         2,670  90    1000     1,499    64         2,971  89    ______________________________________

As shown in Table 5, concentrations of OvIFNτ produced significantantiviral effects. A concentration of only 10 ng/ml resulted in over a50% reduction in RT activity after only six days. A concentration of 500ng/ml resulted in a 90% reduction in RT activity within 10 days.

The viability of human peripheral blood lymphocytes after treatment withIFNτ, over a range of concentrations for 3-13 days, was evaluated bytrypan blue exclusion. The results of this viability analysis arepresented in Table 6.

                  TABLE 6    ______________________________________    Effect of OvIFNτ on Viability of HIV Infected    Human Peripheral Lymphocytes    IFNτ    Concentration               Viable Cells/ml × 10.sup.5    (ng/ml)    Day 3        Day 6   Day 13    ______________________________________    0          16.0         7.5     5.3    10         13.0         7.5     6.0    50         13.0         11.5    9.0    100        15.0         8.5     9.5    500        16.5         12.0    11.0    1000       21.9         9.5     8.5    ______________________________________

The data presented in Table 6 show no evidence of cytotoxic effectsattributable to the administration of IFNτ.

EXAMPLE 13 Inhibition of Cellular Growth

The effects of IFNτ on cellular growth were also examined. Anti-cellulargrowth activity was examined using a colony inhibition assay. Humanamnion (WISH) or MDBK cells were plated at low cell densities to formcolonies originating from single cells. Cells were cultured at 200 or400 cells/well in 24 well plates in HMEM supplemented with 2% fetalbovine serum (FBS) and essential and nonessential amino acids. Variousdilutions of interferons were added to triplicate wells, and the plateswere incubated for 8 days to allow colony formation. Colonies werevisualized after staining with crystal violet, and counted. Cell cycleanalysis was performed with HMEM containing 0.5% "spent" media for anadditional 7 days. WISH cells were used without being synchronized.

For examination of IFNτ activity, cells were replated at 2.5×10⁵cells/well in HMEM with 10% FBS in 6 well plates. Various dilutions ofOvIFNτ alone or in combination with peptides were added to achieve afinal volume of 1 ml. Plates were incubated at 37° C. in 5% Co₂ for 12,15, 18, 24, or 48 hours. Cells were treated with trypsin, collected bylow speed centrifugation and washed. The cell pellet was blotted dry and250 μl of nuclear staining solution (5 mg propidium iodide, 0.3 ml NP40and 0.1 gm sodium citrate in 100 ml distilled H₂ O) was added to eachtube. The tubes were incubated at room temperature. After 10 minutes,250 μl of RNase (500 units/ml in 1.12% sodium citrate) was added pertube and incubated an additional 20 minutes. Nuclei were filteredthrough 44 μm mesh, and analyzed on a FACStar (Becton Dickinson,Mountain View, Calif.) using the DNA Star 2.0 software.

In the cellular growth assay using colony formation of both the bovineepithelial line, MDBK, and the human amniotic line, WISH, OvIFNτinhibited both colony size and number. Ovine IFNτ was more effectivethan human IFNα on the human cell line; thus, it is very potent incross-species activity. Its activity was dose-dependent, and inhibitionof proliferation could be observed at concentrations as low as 1unit/ml. Concentrations as high as 50,000 units/ml (units of antiviralactivity/ml) stopped proliferation, while cell viability was notimpaired.

Cell cycle analysis by flow cytometry with propidium iodide-stained WISHcells revealed an increased proportion of cells in G2/M after 48 hoursof OvIFNτ treatment. IFNτ, therefore, appears to inhibit progress ofcells through S phase. Ovine IFNτ antiproliferative effects can beobserved as early as 12 hours after the initiation of culture and aremaintained through 6 days.

The results presented above demonstrate both the antiproliferativeeffect of IFNτ as well as its low cytotoxicity.

EXAMPLE 14 Further Antiproliferative Effects of IFNτ

The antiproliferative effects of OvIFNτ were studied for a rat cell lineand a bovine cell line. The rate of ³ H-thymidine incorporation was usedto assess the rate of cellular proliferation.

Rat (MtBr7 .c5) or bovine kidney (MDBK) cells were seeded in phenolred-free DME-F12 medium supplemented with 3% dextran-coated charcoalstripped Controlled Process Serum Replacement 2 (CPSR 2, Sigma) and 5%dextran-coated charcoal stripped fetal bovine serum (FBS). Afterattaching for approximately 15-18 hours, the cells were washed once withserum-free DME-F12 medium. The medium was replaced with phenol red-freeDME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS("3/1" medium) or 3/1 medium containing OvIFNτ at various units ofantiviral activity as determined in the vesicular stomatitis viruschallenge assay for interferons (Example 2). Media containing a similardilution of buffer (undiluted buffer=10 mM Tris, 330 mM NaCl, TS!), inwhich the OvIFNτ was dissolved was used for controls.

Cells were pulse labeled with ³ H-thymidine for 2 hours at approximately48 hours post-treatment. The trichloroacetic acid (TCA) precipitableincorporated counts were determined by scintillation counting. Threereplicates were included per treatment. Mean values for OvIFNτtreatments were compared to samples containing comparable dilutions ofcarrier TS buffer. Results of these experiments are shown in Table 7.

                  TABLE 7    ______________________________________    .sup.3 H-Thymidine Incorporation                  % Reduction .sup.3 H-Thymidine    Treatment     Incorporation    ______________________________________    Experiment 1: MtBr7 .c5 (Rat)    3/1           --    10.sup.3 u OvIFNτ/ml 0        (+12)    1:5000 TS     --    10.sup.4 u OvIFNτ/ml 24    1:500 TS      --    10.sup.5 u OvIFNτ/ml 87    Experiment 2: MDBK    3/1           --    10.sup.3 u OvIFNτ/ml 74    1:5000 TS     --    10.sup.4 u OvIFNτ/ml 83    1:500 TS      --    10.sup.5 u OvIFNτ/ml 83    ______________________________________

As can be seen from Table 7, OvIFNτ drastically reduced the rate ofcellular proliferation (based on thymidine incorporation) for each ofthe cell lines tested.

EXAMPLE 15 Antiproliferative Effects of IFNτ on Human Tumor Cell Lines

The antiproliferative activity of OvIFNτ on human tumor cell lines wasevaluated by measuring the rate of ³ H-thymidine incorporation intocells which have been treated with OvIFNτ.

For experiments on tumor lines that grow in suspension, 1 ml of cellswere plated at from 2.5-5×10⁵ cells/well in 24-well plates. Triplicatewells received either the appropriate media, 100, 1,000 or 10,000units/ml of OvIFNτ or equivalent antiviral concentrations of rHuIFNα2A(Lee Biomolecular). After 48 hours of incubation, cells were counted andviability assessed by trypan blue exclusion.

Adherent tumor lines were plated at 2.5×10⁵ cells/well in 1 ml in 6-wellplates. They received interferon treatments as just described, but weretrypsinized prior to counting.

Significant differences between treatments were assessed by an analysisof variance followed by Scheffe's F-test. Cell cycle analysis wasperformed by flow cytometry using propidium iodide.

A. Breast Adenocarcinoma Cells

Human MCF7 breast adenocarcinoma cells were seeded from logarithmicallygrowing cultures in phenol red-free DME-F12 medium supplemented with 3%dextran-coated charcoal stripped CPSR and 5% dextran-coated FBS. Afterattaching for approximately 15-18 hours, the cells were washed once withserum-free DME-F12 medium. The medium was replaced with phenol red-freeDME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS("3/1" medium) or 3/1 medium containing OvIFNτ at the indicated numberof units of antiviral activity as determined in the vesicular stomatitisvirus challenge assay for interferons. Media containing a similardilution of buffer (undiluted buffer=10 mM Tris, 330 mM NaCl TS!) wasused for controls. Cells were pulse labeled with ³ H-thymidine for 2hours at approximately 48 hours post-treatment.

The trichloroacetic acid (TCA) precipitable incorporated counts weredetermined by scintillation counting. Three replicates were included pertreatment. Mean values for OvIFNτ treatments were compared to samplescontaining comparable dilutions of carrier TS buffer. The results ofthese analyses are shown in Table 8.

                  TABLE 8    ______________________________________    .sup.3 H-Thymidine Incorporation                  % Reduction .sup.3 H-Thymidine    Treatment     Incorporation    ______________________________________    MCF7 Human    3/1           --    10.sup.3 u OvIFNτ/ml                  35    1:5000 TS     --    10.sup.4 u OvIFNτ/ml                  53    1:500 TS      --    10.sup.5 u OvIFNτ/ml                  70    ______________________________________

As can be seen from the results shown in Table 8, OvIFNτ was able tosubstantially reduce the rate of ³ H-thymidine incorporation in thehuman carcinoma cell line. This demonstrates the efficacy of OvIFNτ ininhibiting tumor cell proliferation, in particular, mammary tumor cellproliferation.

B. Human Promyelocytic Leukemia

A comparison of the antiproliferative effects of OvIFNτ and IFNα wasconducted using HL-60 (human leukemia) cells (Foa, et al.; Todd, et al.)essentially as described above for MDBK cells. Both OvIFNτ and rHuIFNαinhibit HL-60 cell proliferation. Results of one of three replicateexperiments are presented as mean % growth reduction±SD in FIG. 4. FIG.4 shows that both OvIFNτ and IFNα were able to drastically reduce growthof HL-60 cells. The growth reduction for each compound exceeded 60% foreach concentration tested. At 10,000 units/ml, OvIFNτ caused anapproximately 80% reduction in growth while IFNα caused a 100% reductionin growth.

However, the data presented in FIG. 4 reveal, that a substantial factorin the ability of IFNα to reduce growth was its toxic effect on thecells. At 10,000 units/ml, the toxicity of IFNα resulted in less than25% of the cells remaining viable. By contrast, nearly 100% of the cellsremained viable when OvIFNτ was applied at 10,000 units/ml.

FIG. 5 presents data demonstrating that rHuIFNα is cytotoxic. In thefigure, results of one of three replicate experiments are presented asmean % viability±SD.

C. Human Cutaneous T Cell Lymphoma

The cutaneous T cell lymphoma, HUT 78, responded similarly to HL-60 whentreated with IFNτ (FIG. 9). Both OvIFNτ and rHuIFNα reduce HUT 78 cellgrowth, but 10,000 units/ml of rHuIFNα decreased the cell number belowthat originally plated (5×10⁵). This is indicative of a reduction incell viability to approximately 60%.

Cell cycle analysis (performed by cell flow cytometry) revealed anincreased proportion of cells in G2/M phase of the cell cycle upon 48hours of treatment with both interferons (Table 9). In Table 9 theresults from one of three replicate experiments are presented as thepercentage of cells in each phase of the cell cycle. 10,000 events wereanalyzed per sample.

This result is likely due to the slower progress of cells through thecell cycle. In the sample treated with 10,000 units/ml of rHuIFNα, alarge percentage of events with low forward and high side scatter,identifying dead cells, were present. This is consistent with the dataobtained from proliferation experiments, where only OvIFNτ inhibited HUT78 proliferation without toxicity.

                  TABLE 9    ______________________________________    HUT 78 Cell Cycle Analysis    Treatment    (units/ml)    G0/G1      S      G2/M    ______________________________________    Media         44.43      49.95  5.61    100          OvIFNτ                          44.35    47.45                                        8.20    100          rHuIFNα                          40.01    57.53                                        2.45    1,000        OvIFNτ                          41.29    50.50                                        8.21    1,000        rHuIFNα                          41.73    44.91                                        13.36    10,000       OvIFNτ                          42.79    42.61                                        14.60    10,000       rHuIFNα                          18.01    71.31                                        10.67                                        (cell death)    ______________________________________

D. Human T Cell Lymphoma

The T cell lymphoma cell line H9 was slightly less sensitive to theantiproliferative effects of the IFNs than the tumor cell linesdescribed above. Results of one of three replicate experiments arepresented in FIG. 10 as mean % growth reduction±SD. While rHuIFNα wasnot toxic to the H9 cells, it failed to inhibit cell divisionsignificantly at any of the concentrations examined. In contrast, OvIFNτwas observed to reduce H9 growth by approximately 60% (FIG. 10). Thus,only OvIFNτ is an effective growth inhibitor of this T cell lymphoma.

The results presented above demonstrate both the antiproliferativeeffect of IFNτ as well as its low cytotoxicity.

EXAMPLE 16 Preliminary In Vivo Treatment with OvIFNτ

Three groups of 4 C57Bl/6 mice per group were given 2.5×10⁴ B16-F10cells via the tail vein: B16-F10 is a syngeneic mouse transplantabletumor selected because of its high incidence of pulmonary metastases(Poste, et al., 1981). Interferon treatment was initiated 3 days afterthe introduction of the tumor cells. Each mouse received 100 μl ofeither PBS alone, PBS containing 1×10⁵ units of OvIFNτ , or PBScontaining 1×10⁵ units of recombinant murine IFNα (MuIFNα), i.v. per dayfor 3 consecutive days.

Mice were sacrificed at 21 days and the lungs were preserved in 10%buffered formalin. The frequency of pulmonary metastases were comparedbetween control mice (PBS), OvIFNτ-treated mice, and MuIFNα-treatedmice. The results of these in vivo administrations demonstrated thatOvIFNτ dramatically reduced B16-F10 pulmonary tumors. These resultssupport the use of IFNτ as an efficacious antineoplastic agent in vivo.

EXAMPLE 17 Competitive Binding of IFNτ Peptide Fragments

A. The Ability of IFNτ-Based Peptides to Block IFNτ and IFN-α AntiviralActivity

Overlapping synthetic peptides were synthesized corresponding to theentire IFNτ sequence (FIG. 6). Average hydropathicity values werecalculated by taking the sum of the hydropathy values for each aminoacid divided by the total number of amino acids in each sequence.Hydropathy values were taken from Kyte, et al. (1982).

These peptides were of approximately the same molecular weight butdiffered slightly in overall hydrophilicity. Despite this difference,all peptides were antigenic as demonstrated by the production of rabbitantisera with titers greater than 1:3,000 as assessed by ELISA (Harlow,et al.).

The peptides were used to inhibit the antiviral activity (Example 2) ofOvIFNτ and rBoIFNα. The results of this analysis are presented in FIG.12: 1 mM N- and C-terminal peptides both effectively blocked theantiviral activity of OvIFNτ using MDBK cells. A third peptide,representing amino acids 62-92, also reduced IFNτ antiviral activity(70% inhibition). The peptide OvIFNτ (119-150) showed minimal inhibitoryactivity. The OvIFNτ (34-64) and (90-122) peptides had no apparentinhibitory activity.

Peptide inhibition of OvIFNτ antiviral activity was also examined asfollows. Monolayers of Madin Darby bovine kidney cells were incubatedwith 40 units/ml OvIFNτ in the presence or absence of variousconcentrations of OvIFNτ peptides (see FIG. 13). Results in FIG. 13 areexpressed as the percent of control antiviral activity: that is, in theabsence of any competing peptide. Data presented are the means of 6replicate experiments. The data demonstrate that inhibition by OvIFNτ(1-37), (62-92), (119-150), and (139-172) were significantly differentthan OvIFNτ (34-64) and (90-122) at 10⁻³ M and 3×10⁻³ M. OvIFNτ(139-172) was significantly different than all other peptides at 10⁻³ M.Significance was assessed by analysis of variance followed by Scheffe'sF test at p<0.05. Thus, OvIFNτ (1-37) (62-92), (119-150), and (139-172),in particular (139-172), may represent receptor binding regions forIFNτ.

The ability of the OvIFNτ peptides to inhibit bovine IFNα (BoIFNα)antiviral activity was examined as follows. Monolayers of Madin Darbybovine kidney cells were incubated with 40 units/ml bovine IFNα in thepresence or absence of various concentrations of OvIFNτ peptides. Theresults are presented in FIG. 14 and are expressed as the percent ofcontrol antiviral activity in the absence of OvIFNτ peptides. The datapresented are the means of 4 replicate experiments. The results indicatethat inhibition by OvIFNτ (62-92), (119-150), and (13914 172) weresignificantly different from OvIFNτ (1-37), (34-64) and (90-122) at 10⁻³M. OvIFNτ (139-172) was significantly different than OvIFNτ (1-37),(34-64) and (90-122) at 3×10⁻³ M. Significance was assessed by analysisof variance followed by Scheffe's F test at p<0.05. Thus, OvIFNτ(62-92), (119-150), and (139-172), in particular (139-172), mayrepresent common receptor binding regions for IFNτ and bovine IFNα.

Peptide inhibition by OvIFNτ peptides of human IFNα antiviral activitywas also examined. Monolayers of Madin Darby bovine kidney cells wereincubated with 40 units/ml human IFNα in the presence or absence ofvarious concentrations of OvIFNτ peptides. The results are expressed asthe percent of control antiviral activity in the absence of OvIFNτpeptides. The data are presented in FIG. 15 and are the means of 3replicate experiments. OvIFNτ (139-172) was significantly different fromall other peptides at 10⁻³ M. Significance was assessed by analysis ofvariance followed by Scheffe's F test at p<0.05. Thus, OvIFNτ (139-172)may represent a common receptor binding region for IFNτ and variousIFNα(s).

The OvIFNτ peptides described above appear to have no effect on theantiviral activity of IFNγ. Peptide inhibition of bovine IFNγ antiviralactivity was evaluated as follows. Monolayers of Madin Darby bovinekidney cells were incubated with 40 units/ml bovine IFN gamma in thepresence or absence of various concentrations of OvIFNτ peptides.Results are expressed as the percent of control antiviral activity inthe absence of OvIFNτ peptides. The data are presented in FIG. 16 andare the means of 3 replicate experiments. There were no significantdifferences among peptides as assessed by analysis of variance followedby Scheffe's F test at p<0.05.

The two synthetic peptides OvIFNτ (1-37) and OvIFNτ (139-172) alsoblocked OvIFNτ anti-FIV and anti-HIV activity. Reverse transcriptase(RT) activity (Examples 12 and 13) was monitored over a 14 day period inFIV-infected FET-1 cells (1×10⁶ /ml) and HIV-infected HPBL (1×10⁶ /ml).Control cultures received no OvIFNτ. OvIFNτ was used at 100 ng/ml, andpeptides were used at 200 μM. Data from a representative experiment areexpressed as cpm/ml culture supernatant and are presented for FIVinfected cells, FIG. 11A, and HIV infected cells, FIG. 11B. Both the N-and C-terminus of OvIFNτ appear to be involved in its anti-retroviralactivity. While both peptides blocked FIV RT activity, only theC-terminal peptide, OvIFNτ(139-172), was an efficient inhibitor ofvesicular stomatitis virus activity on the feline cell line, Fc9. Thusthe C-terminal regions of type I IFNs may bind to common site on thetype I IFN receptor, while the N-terminal region may be involved in theelicitation of unique functions.

B. Anti-Peptide Sera

The ability of anti-peptide antisera to inhibit OvIFNτ antiviralactivity was also determined. Antipeptide antisera inhibition of OvIFNτantiviral activity was evaluated as follows. Monolayers of MDBK cellswere incubated with 20 units/ml of OvIFNτ in the presence a 1:30dilution of either preimmune sera or antisera to each of the OvIFNτpeptides described above. In FIG. 17 the data from duplicate experimentsare presented as the mean percent inhibition of OvIFNτ antiviralactivity produced by antipeptide antisera relative to the appropriatepreimmune sera±standard error. Significant differences were assessed byanalysis of variance followed by Scheffe's F test at p<0.05. Consistentwith peptide inhibition of antiviral activities, sera containingantibodies immunoreactive to OvIFNτ (1-37), OvIFNτ (62-92), and OvIFNτ(139-172) were also the most effective inhibitors of OvIFNτ antiviralactivity, with antibodies directed against the N-terminal and C-terminalpeptides being the most efficacious.

The same sera were also used to examine their effect on the binding ofIFNτ to its receptor.

The IFNτ binding assay was carried out as follows. Five μg of IFNτ wasiodinated for 2 minutes with 500 μCi of Na¹²⁵ I (15 mCi/μg; AmershamCorporation, Arlington Heights, Ill.) in 25 μl of 0.5M potassiumphosphate buffer, pH 7.4, and 10 μl of chloramine-T (5 mg/ml) (Griggs,et al., 1992). The specific activity of the iodinated protein was 137μCi/μg. For binding assays, monolayers of MDBK cells were fixed withparaformaldehyde and blocked with 5% nonfat dry milk. Cells wereincubated with 5 nM ¹²⁵ I-IFNτ in phosphate buffered saline with 1% BSAfor 2 hours at 4° C. in the presence or absence of a 1:30 dilution ofsera containing antibodies raised against IFNτ peptides or theappropriate preimmune sera. Specific binding was assessed by incubationwith a 100-fold molar excess of unlabeled IFNτ. Specific binding of 36%was determined by competition with 500 nM unlabeled IFNτ. For example,total counts bound were 6850±133, and a 100-fold molar excess of OvIFNτproduced 4398±158 counts per minute. After incubation, the monolayerswere washed three times, solubilized with 1% sodium dodecyl sulfate, andthe radioactivity counted. Data from three replicate experiments arepresented in FIG. 18 as the mean percent reduction of OvIFNτ specificbinding produced by antipeptide antisera relative to the appropriatepreimmune sera±standard deviation. Significant differences were assessedby analysis of variance followed by Scheffe's F test.

The same sera (containing antibodies immunoreactive to OvIFNτ (1-37),OvIFNτ (62-92), and OvIFNτ (139-172)) were the most effective inhibitorsof ¹²⁵ I-IFNτ binding to its receptor on MDBK cells. The lack of effectof sera immunoreactive with other IFNτ-derived peptides was not afunction of titer against OvIFNτ, since each sera had equal or greatertiter to their respective peptide relative to the three inhibiting sera:similar results were obtained when sera reactivity against the wholeOvIFNτ molecule was assessed by ELISA for each sera.

These peptides, although apparently binding to the interferon receptor,did not in and of themselves elicit interferon-like effects in thecells.

C. Anti-Proliferative Activity

Functionally important sites for the antiproliferative activity of IFNτwere also examined using synthetic peptides (Table 10). Cellularproliferation was assayed as described above using MDBK cells. MDBKcells were cultured at 5×10⁵ cells/well in experiments 1 and 2 or 10×10⁵cells in experiment 3 and treated with medium alone, IFNτ at aconcentration of 300 units/ml and peptides at 1 mM for 48 hours.Duplicate wells were counted in each of three replicate experiments. Forstatistical analysis, data were normalized based on medium alone andassessed by analysis of variance followed by Least SignificantDifference multiplate comparison test (p>0.05).

                                      TABLE 10    __________________________________________________________________________    Peptide Inhibition of IFNτ Antiproliferative Activity               Experiment 1                        Experiment 2                                Experiment 3               Cell Via-                        Cell Via-                                Cell Via-    Treatment  Count                    bility                        Count                             bility                                Count                                     bility    __________________________________________________________________________    Medium alone               9.8 × 10.sup.5                    99% 13.0 × 10.sup.5                             96%                                27.3 × 10.sup.5                                     97%    IFNτ   5.0 × 10.sup.5                    98% 5.6 × 10.sup.5                             97%                                8.3 × 10.sup.5                                     97%    IFNτ + IFNτ (1-37)               6.3 × 10.sup.5                    100%                        10.6 × 10.sup.5                             98%                                13.4 × 10.sup.5                                     100%    IFNτ + IFNτ (34-64)               5.3 × 10.sup.5                    96% 6.9 × 10.sup.5                             95%                                16.0 × 10.sup.5                                     98%    IFNτ + IFNτ (62-92)               6.5 × 10.sup.5                    97% 9.2 × 10.sup.5                             93%                                8.9 × 10.sup.5                                     96%    IFNτ + IFNτ (90-122)               5.9 × 10.sup.5                    100%                        11.0 × 10.sup.5                             97%                                19.6 × 10.sup.5                                     98%    IFNτ + IFNτ (119-150)               8.4 × 10.sup.5                    100%                        13.2 × 10.sup.5                             96%                                31.8 × 10.sup.5                                     90%    IFNτ + IFNτ (139-172)               5.1 × 10.sup.5                    100%                        12.7 × 10.sup.5                             98%                                18.9 × 10.sup.5                                     98%    __________________________________________________________________________

When proliferation of MDBK cells was monitored over a two-day period,cell number increased roughly 2-fold with greater than 95% viability.Addition of 300 units/ml of OvIFNτ entirely eliminated cellproliferation without a decrease in cell viability. Ovine IFNτ (119-150)was the most effective inhibitor of IFNτ antiproliferative activity.

Antisera to IFNτ (119-150), which inhibited binding of OvIFNτ toreceptor, also reversed the OvIFNτ antiproliferative effect. Severalother peptides, notably IFNτ (139-172), reversed the OvIFNτantiproliferative effect, but to a lesser extent.

EXAMPLE 18 Further Analysis of the Cellular and Anti-Viral Effects ofIFNτ

A. HIV Anti-Viral Effects

The antiviral effects of IFNτ against HIV were evaluated by treatinghuman PBMC cells with various amounts of either recombinant ovine IFNτ(r-OvIFNτ ) or recombinant human IFNα2a at the time of infection withHIV. IFNτ was present throughout the experiment. At day 7 and day 14,p24 production was determined (by ELISA (Wang, et al., 1988, 1989) andcompared to a zero drug control. The results of this analysis arepresented in Table 11.

                  TABLE 11    ______________________________________    Amounts of Drug       %        %    Units/ml              Inhibition                                   Inhibition    IFNα2a             IFNτ     Day 7    Day 14    ______________________________________    10                    58%, 48% 91%, 91%             26           48%, 45% 88%, 59%    100                   68%, 74% 94%, 91%             260          58%, 51% 82%, 70%    1,000                 89%, 86% 97%, 93%             2,600        65%, 68% 87%, 79%    10,000                90%, 86% 99%, 99%             26,000       77%, 85% 77%, 96%             260,000      85%, 84% 96%, 86%    ______________________________________

The data from these experiments support the conclusion that, atrelatively low concentrations, IFNα2a and IFNτ are effective in reducingthe replication of HIV in human lymphocytes.

B. In vitro Cytotoxicity Test in PBMC's

Human PBMC's were seeded at 5×10⁵ cells/ml. Cells were stimulated at day0 with 3 μg/ml PHA. Cells were treated with recombinant human IFNα2A (atconcentrations of 10, 100, 1,000 and 10,000 units/ml) and IFNτ (atconcentrations of 2.6, 26, 260, 2,600, 26,000, 260,000, and 2,600,000units/ml) in 200 μl/wells (4 replicates of each concentration using 96well flat bottom plates). Control cultures were given no interferons.After 4 days of incubation, cells were pulsed for 9 hours using ³H-thymidine at 1 uCi/well. The cells were harvested and theincorporation of labeled thymidine into DNA was determined (FIG. 8).

No cytotoxicity was observed by measuring the uptake of thymidine at anyconcentration of IFNτ. However, rHuIFNα2 was toxic to cells at 1,000units/ml.

In a second experiment, the same human PBMC's were treated with eitherIFNτ or human IFNα2A at concentrations of 100 units/ml or 10,000units/ml. After 3 days or 8 days of incubation, viable cells werecounted by flow cytometry. The results of this analysis are presented inTable 12.

                  TABLE 12    ______________________________________                     Number of Viable                     Cells × 10,000    Treatment (units/ml)                       Day 3   Day 8    ______________________________________    No treatment       735     840    IFNτ 100    units/ml   745   860    IFNτ 10,000 units/ml   695   910    IFNα2a 100                    units/ml   635   750    IFNα2a 10,000                    units/ml   680   495    ______________________________________

No cytotoxicity was observed in the cells treated with IFNτ. However,there was 10% cell death in IFNα2a treated cells at Day 3 and 49% celldeath at Day 8.

C. Inhibition of Hepatitis B Virus DNA Replication in Hepatocytes

The cell line used, HepG2-T14, is a human cell that was derived fromliver cells transfected with Hepatitis B Virus (HBV). The cell linesemi-stably produces HBV virus: over time the cell line's production ofHBV intracellular DNA and secreted virus decreases. In order to maximizeproduction of HBV DNA and virus, the cells are pre-treated with deAZA-C(5-azacytidine; Miyoshi, et al.) to induce production of the virus.Treatment was for 2-3 days and the amount of induction was about afactor of two.

The cells were then treated with either the IFNα and IFNτ at levels of0, 5,000, 10,000, 20,000 and 40,000 units per ml.

All levels of either IFNα or IFNτ reduced DNA production by about afactor of 2 compared to the no drug control.

D. Inhibition of Hepatospecific Messenger RNA Production in Hepatocytes

The hepatocyte cell line HepG2-T14 (described above) was examined forthe effects of IFNα and IFNτ on hepatospecific mRNA production. Cellswere incubated in concentrations of IFNα or IFNτ at 0, 5,000, 10,000,20,000, and 40,000 units per ml. The messenger RNAs for the hepatocytespecific proteins Apo E and Apo A1 were detected by hybridizationanalysis (Sambrook, et al.; Maniatis, et al.) using probes specific forthese two mRNA's (Shoulders, et al., and Wallis, et al.).

No reduction of mRNA production was seen for Apo E or Apo A1 mRNAproduction with up to 40,000 units of either IFNα or IFNτ. This resultsuggests that the reduction of viral DNA replication in previousexperiments was not due to the effects of IFNs on cellular house-keepingactivities; rather the reduction was likely due to specific inhibitionof viral replication in the host cells.

E. In Vitro Toxicity of IFNβ, IFNγ and IFNτ-L929 Cell Assay

The toxicity of IFN treatment was measured in vitro using the mouse L929cell line. L929 cells were treated with 6000 U/ml to 200,000 U/ml ofeither OvIFNτ or MuIFNβ. The interferons were added at time zero and thecells were incubated for 72 hours and stained with crystal violet. Thepercentage of living cells was determined by measuring the absorbance at405 nm.

Exemplary data are shown in FIG. 21. Values are presented as percentviability±standard error in which 100 percent is equal to the viabilityof L929 cells treated with media alone. At 6000 U/ml, IFNβ-treated cellsexhibited a 77.0±0.6% viability. Viability of L929 cells decreased asthe concentrations of IFNβ increased in a dose-dependent manner. Incontrast, L929 cells showed no decrease in viability at any of the IFNτconcentrations tested. These data indicate that, unlike IFNβ, IFNτ lackstoxicity at high concentrations in vitro.

Taken together, the results summarized above demonstrate that IFNτ isessentially non-toxic at concentrations at which IFNβ induces toxicityboth in vitro and in vivo.

F. In Vivo Toxicity of IFNβ, IFNγ and IFNτ--Cell Counts and WeightChanges

The effects of in vivo treatment with IFNτ, IFNβ and IFNα (10⁵U/injection) on total white blood cell (WBC), total lymphocyte countsand weight measurements in NZW mice were assessed as follows.Interferons (OvIFNτ, MuIFNβ, and MuIFNα) were injected intraperitoneally(i.p.) at a concentration of 10⁵ U in a total volume of 0.2 ml in PBSinto groups of New Zealand White (NZW) mice (Jackson Laboratories, BarHarbor, Me.). Three to four animals were included in each group. Whiteblood cell (WBC) counts were determined before injection and at selectedtimepoints thereafter (typically 12 and 24 hours) using a hemocytometerand standard techniques. Differential WBC counts were performed onWright-Giemsa stained blood smears. The Before injection, the weights ofthe animals ranged from 20 to 23 grams.

The results are summarized in Table 13, below.

                                      TABLE 13    __________________________________________________________________________    IN VIVO TOXICITY OF INTERFERONS AS MEASURED    BY WHITE BLOOD CELL COUNTS AND PERCENT WEIGHT CHANGE                                       % Weight    Cell Count (Cell No. × 10.sup.3)                                 %     Change 24    Before Injection                    12 hr. after Injection                                 Lymphocyte                                       Hours after    IFN       Total WBC             Lymphocytes                    Total WBC                          Lymphocytes                                 Depression                                       Injection    __________________________________________________________________________    none       7.3 ± 1.0             6.4 ± 0.7                    8.0 ± 0.8                          7.1 ± 0.7                                 0     +0.5 ± 0.7    τ       6.7 ± 0.7             5.9 ± 0.6                    6.7 ± 0.5                          5.8 ± 0.4                                 1.7   +1.3 ± 0.5    β       7.0 ± 1.4             6.0 ± 0.5                    6.8 ± 0.8                          4.1 ± 0.3                                 31.7  -20.0 ± 1.0    α       6.0 ± 0.8             5.2 ± 0.7                    4.8 ± 0.5                          2.3 ± 0.2                                 55.8  -8.5 ± 2.0    __________________________________________________________________________

No significant differences in WBC counts, lymphocyte counts or weightchange were observed between IFNτ-treated and untreated mice. Incontrast, IFNβ-treated mice exhibited a 31.7% depression in lymphocytecounts 12 hours after injection, which continued for at least the next12 hours. IFNα-treated mice exhibited a 55.8% lymphocyte depression andsignificant weight loss 12 hours after injection. These data indicatethat, unlike IFNβ and IFNα, IFNτ lacks toxicity in vivo at the aboveconcentrations as evidenced by peripheral blood cell counts and weightmeasurements.

EXAMPLE 19 Isolation of Interferon-τ Fusion Protein

Sepharose 4B beads conjugated with anti-beta galactosidase is purchasedfrom Promega. The beads are packed in 2 ml column and washedsuccessively with phosphate-buffered saline with 0.02% sodium azide and10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin).

The IFNτ coding sequence (e.g., SEQ ID NO:33, i.e., minus thenucleotides corresponding to the leader sequence) is cloned into thepolylinker site of lambda gt11. The IFNτ coding sequence is placedin-frame with the amino terminal β-galactosidase coding sequences inlambda gt11. Lysogens infected with gt11/IFNτ are used to inoculate 500ml of NZYDT broth. The culture is incubated at 32° C. with aeration toan O.D. of about 0.2 to 0.4, then brought to 43° C. quickly in a 43° C.water bath for 15 minutes to induce gt11 peptide synthesis, andincubated further at 37° C. for 1 hour. The cells are pelleted bycentrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4containing 2% "TRITON X-100" and 1% aprotinin added just before use.

The resuspended cells are frozen in liquid nitrogen then thawed,resulting in substantially complete cell lysis. The lysate is treatedwith DNaseI to digest bacterial and phage DNA, as evidenced by a gradualloss of viscosity in the lysate. Non-solubilized material is removed bycentrifugation.

The clarified lysate material is loaded on the Sepharose column, theends of the column closed, and the column placed on a rotary shaker for2 hrs. at room temperature and 16 hours at 4° C. After the columnsettles, it is washed with 10 ml of TX buffer. The fused protein iseluted with 0.1M carbonate/bicarbonate buffer, pH10. Typically, 14 ml ofthe elution buffer is passed through the column, and the fusion proteinis eluted in the first 4-6 ml of eluate.

The eluate containing the fusion protein is concentrated in"CENTRICON-30" cartridges (Amicon, Danvers, Mass.). The final proteinconcentrate is resuspended in, for example, 400 μl PBS buffer. Proteinpurity is analyzed by SDS-PAGE.

For polyclonal antibodies, the purified fused protein is injectedsubcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg offused protein is injected at days 0 and 21, and rabbit serum istypically collected at 6 and 8 weeks.

EXAMPLE 20 Preparation of Anti-IFNτ Antibody

A. Expression of Glutathione-S-Transferase Fusion Proteins

The IFNτ coding sequence (e.g., SEQ ID NO:33) is cloned into the pGEXvector (Boyer, et al.; Frangioni, et al.; Guan, et al.; Hakes, et al.;Smith, et al., 1988). The pGEX vector (Smith, et al.) was modified byinsertion of a thrombin cleavage sequence in-frame with theglutathione-S-transferase protein (GST--sj26 coding sequence). Thisvector is designated pGEXthr. The IFNτ coding sequence is placedin-frame with the sj26-thrombin coding sequences (Guan, et al.; Hakes,et al.). The IFNτ coding sequence insert can be generated by thepolymerase chain reaction using PCR primers specific for the insert.

The IFNτ fragment is ligated to the linearized pGEXthr vector. Theligation mixture is transformed into E. coli and ampicillin resistantcolonies are selected. Plasmids are isolated from the ampicillinresistant colonies and analyzed by restriction enzyme digestion toidentify clones containing the IFNτ insert (vector designatedpGEXthr-IFNτ).

E. coli strain XL-I Blue is transformed with pGEXthr-IFNτ and is grownat 37° C. overnight. DNA is prepared from randomly-picked colonies. Thepresence of the insert coding sequence is typically confirmed by (i)restriction digest mapping, (ii) hybridization screening using labelledIFNτ probes (i.e., Southern analysis), or (iii) direct DNA sequenceanalysis.

B. Partial Purification of Fusion Proteins

A pGEXthr-IFNτ clone is grown overnight. The overnight culture isdiluted 1:10 with LB medium containing ampicillin and grown for one hourat 37° C. Alternatively, the overnight culture is diluted 1:100 andgrown to OD of 0.5-1.0 before addition of IPTG(isopropylthio-β-galactoside). IPTG (GIBCO-BRL, Gaithersburg Md.) isadded to a final concentration of 0.2-0.5 mM for the induction ofprotein expression and the incubation is typically continued for 2-5hours, preferably 3.5 hours.

Bacterial cells are harvested by centrifugation and resuspended in 1/100culture volume of MTPBS (150 mM NaCl, 16 mM Na₂ HPO₄, 4 mM NaH₂ PO₄).Cells are lysed by lysozyme, sonication or French press, and lysatescleared of cellular debris by centrifugation.

An aliquot of the supernatant obtained from IPTG-induced cultures ofpGEXthr-IFNτ-containing cells and an aliquot of the supernatant obtainedfrom IPTG-induced cultures of pGEXthr-vector alone are analyzed bySDS-polyacrylamide gel electrophoresis followed by Western blotting, asdescribed below.

If necessary, the extracts can be concentrated by ultrafiltration using,for example, a "CENTRICON 10" filter.

Alternatively, the fusion proteins are partially purified over aglutathione agarose affinity column as described in detail by Smith, etal. In this method, 100 ml cultures are grown overnight. The culturesare diluted to 1 liter, and the cells grown another hour at 37° C.Expression of the fusion proteins is induced using IPTG. The inducedcultures are grown at 37° C. for 3.5 hours. Cells are harvested and asonicator used to lyse the cells. Cellular debris is pelleted and theclear lysate loaded onto a glutathione "SEPHAROSE" column. The column iswashed with several column volumes. The fusion protein is eluted fromthe affinity column with reduced glutathione and dialyzed. The IFNτ canbe liberated from the hybrid protein by treatment with thrombin. Thesj26 and IFNτ fragments of the hybrid protein can then be separated bysize fractionation over columns or on gels.

Alternatively, the IFNτ portion of the hybrid protein is released fromthe column by treatment with thrombin (Guan, et al.; Hakes, et al.).

C. Antibodies Against the Fusion Protein

The purified Sj26/IFNτ fused protein is injected subcutaneously inFreund's adjuvant in a rabbit. Approximately 1 mg of fused protein isinjected at days 0 and 21, and rabbit serum is typically collected at 6and 8 weeks. A second rabbit is similarly immunized with purified Sj26protein obtained from control bacterial lysate.

Minilysates from the following bacterial cultures are prepared: (1)KM392 cells infected with pGEXthr and pGEXthr containing the IFNτinsert; and (2) cells infected with lambda gt11 containing the IFNτinsert. The minilysates and a commercial source β-galactosidase arefractionated by SDS-PAGE, and the bands transferred to nitrocellulosefilters for Western blotting (Sambrook, et al.; Ausubel, et al.).

Summarizing the expected results, serum from control (Sj26 ) rabbits isimmunoreactive with each of the Sj26 and Sj26 fused protein antigens.Serum from the animal immunized with Sj26/IFNτ fused protein is reactivewith all Sj-26 and beta-gal fusion proteins containing IFNτ codingsequences, indicating the presence of specific immunoreaction with theIFNτ antigen. None of the sera are expected to be immunoreactive withbeta-galactosidase.

Anti-IFNτ antibody present in the sera from the animal immunized withthe Sj26/IFNτ is purified by affinity chromatography (using immobilizedrecombinantly produced IFNτ as ligand, essentially as described above inExample 12 for the anti-beta-galactosidase antibody).

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 44    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 516 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: circular    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: Ovis arie - #s              (B) STRAIN: Domestic    #Blastula (blastocyst)ENTAL STAGE:              (F) TISSUE TYPE: Trophe - #ctoderm              (G) CELL TYPE: Mononucl - #ear trophectoderm cells    -    (vii) IMMEDIATE SOURCE:              (B) CLONE: oTP-1a    -   (viii) POSITION IN GENOME:              (C) UNITS: bp    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..516    -      (x) PUBLICATION INFORMATION:    #L        (A) AUTHORS: Ott, Troy                   Van Heeke - #, Gino    #Howard M      Johnson,                   Bazer, Fu - #ller W    #Expression in Saccharomyces and    #of a Synthetic Gene for the Type I    #Interferon Ovine Trophoblast                   Protein-1:Pu - #rification and Antiviral Activity              (C) JOURNAL: J. Interfe - #ron Res.              (D) VOLUME: 11              (F) PAGES: 357-364              (G) DATE: 1991              (K) RELEVANT RESIDUES I - #N SEQ ID NO:1: FROM 1 TO 516    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - TGC TAC CTG TCG CGA AAA CTG ATG CTG GAC GC - #T CGA GAA AAT TTA AAA      48    Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Al - #a Arg Glu Asn Leu Lys    #                 15    - CTG CTG GAC CGT ATG AAT CGA TTG TCT CCG CA - #C AGC TGC CTG CAA GAC      96    Leu Leu Asp Arg Met Asn Arg Leu Ser Pro Hi - #s Ser Cys Leu Gln Asp    #             30    - CGG AAA GAC TTC GGT CTG CCG CAG GAA ATG GT - #T GAA GGT GAC CAA CTG     144    Arg Lys Asp Phe Gly Leu Pro Gln Glu Met Va - #l Glu Gly Asp Gln Leu    #         45    - CAA AAA GAC CAA GCT TTC CCG GTA CTG TAT GA - #A ATG CTG CAG CAG TCT     192    Gln Lys Asp Gln Ala Phe Pro Val Leu Tyr Gl - #u Met Leu Gln Gln Ser    #     60    - TTC AAC CTG TTC TAC ACT GAA CAT TCT TCG GC - #C GCT TGG GAC ACT ACT     240    Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - CTT CTA GAA CAA CTG TGC ACT GGT CTG CAA CA - #G CAA CTG GAC CAT CTG     288    Leu Leu Glu Gln Leu Cys Thr Gly Leu Gln Gl - #n Gln Leu Asp His Leu    #                 95    - GAC ACT TGC CGT GGC CAG GTT ATG GGT GAA GA - #A GAC TCT GAA CTG GGT     336    Asp Thr Cys Arg Gly Gln Val Met Gly Glu Gl - #u Asp Ser Glu Leu Gly    #           110    - AAC ATG GAT CCG ATC GTT ACT GTT AAA AAA TA - #T TTC CAG GGT ATC TAC     384    Asn Met Asp Pro Ile Val Thr Val Lys Lys Ty - #r Phe Gln Gly Ile Tyr    #       125    - GAC TAC CTG CAG GAA AAA GGT TAC TCT GAC TG - #C GCT TGG GAA ATC GTA     432    Asp Tyr Leu Gln Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Ile Val    #   140    - CGC GTT GAA ATG ATG CGG GCC CTG ACT GTG TC - #G ACT ACT CTG CAA AAA     480    Arg Val Glu Met Met Arg Ala Leu Thr Val Se - #r Thr Thr Leu Gln Lys    145                 1 - #50                 1 - #55                 1 -    #60    #      516ACT AAA ATG GGT GGT GAC CTG AAT TC - #T CCG    Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 172 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of a mature:    #protein       OvIFNtau    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Al - #a Arg Glu Asn Leu Lys    #                 15    - Leu Leu Asp Arg Met Asn Arg Leu Ser Pro Hi - #s Ser Cys Leu Gln Asp    #             30    - Arg Lys Asp Phe Gly Leu Pro Gln Glu Met Va - #l Glu Gly Asp Gln Leu    #         45    - Gln Lys Asp Gln Ala Phe Pro Val Leu Tyr Gl - #u Met Leu Gln Gln Ser    #     60    - Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - Leu Leu Glu Gln Leu Cys Thr Gly Leu Gln Gl - #n Gln Leu Asp His Leu    #                 95    - Asp Thr Cys Arg Gly Gln Val Met Gly Glu Gl - #u Asp Ser Glu Leu Gly    #           110    - Asn Met Asp Pro Ile Val Thr Val Lys Lys Ty - #r Phe Gln Gly Ile Tyr    #       125    - Asp Tyr Leu Gln Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Ile Val    #   140    - Arg Val Glu Met Met Arg Ala Leu Thr Val Se - #r Thr Thr Leu Gln Lys    145                 1 - #50                 1 - #55                 1 -    #60    - Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 516 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (vi) ORIGINAL SOURCE:    #synthetic nucleotide sequence encoding    #human interferon-tau protein, HuIFNtau1.    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - TGTGACTTGT CTCAAAACCA CGTTTTGGTT GGTAGAAAGA ACTTAAGACT AC - #TAGACGAA      60    - ATGAGACGTC TATCTCCACG CTTCTGTCTA CAAGACAGAA AGGACTTCGC TT - #TGCCTCAG     120    - GAAATGGTTG AAGGTGGCCA ACTACAAGAA GCTCAAGCGA TATCTGTTTT GC - #ACGAAATG     180    - TTGCAACAAA GCTTCAACTT GTTCCACACC GAACACTCTT CGGCCGCTTG GG - #ACACCACC     240    - TTGTTGGAAC AGCTCAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGA TG - #CATGTTTG     300    - GGTCAAGTTA TGGGTGAAGA AGACTCTGCT CTCGGGAGAA CCGGTCCAAC GC - #TAGCTTTG     360    - AAGAGATACT TCCAAGGTAT CCACGTTTAC TTGAAGGAAA AGGGTTACTC TG - #ACTGTGCT     420    - TGGGAAACCG TGCGTCTAGA AATCATGCGT AGCTTCTCTT CTTTGATCAG CT - #TGCAAGAA     480    #      516         ACGG TGACTTGTCG AGCCCA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 172 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence for a mature    #protein, HuIFNtau1.tau    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Arg Lys Asn Leu Arg    #                15    - Leu Leu Asp Glu Met Arg Arg Leu Ser Pro Ar - #g Phe Cys Leu Gln Asp    #            30    - Arg Lys Asp Phe Ala Leu Pro Gln Glu Met Va - #l Glu Gly Gly Gln Leu    #        45    - Gln Glu Ala Gln Ala Ile Ser Val Leu His Gl - #u Met Leu Gln Gln Ser    #    60    - Phe Asn Leu Phe His Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    #80    - Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gl - #n Gln Leu Asp Asn Leu    #                95    - Asp Ala Cys Leu Gly Gln Val Met Gly Glu Gl - #u Asp Ser Ala Leu Gly    #           110    - Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Ty - #r Phe Gln Gly Ile His    #       125    - Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Thr Val    #   140    - Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Le - #u Ile Ser Leu Gln Glu    145                 1 - #50                 1 - #55                 1 -    #60    - Arg Leu Arg Met Met Asp Gly Asp Leu Ser Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment 1-37    #ID NO:2       of SEQ    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Al - #a Arg Glu Asn Leu Lys    #15    - Leu Leu Asp Arg Met Asn Arg Leu Ser Pro Hi - #s Ser Cys Leu Gln Asp    #             30    - Arg Lys Asp Phe Gly             35    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment 34-64    #ID NO:2       of SEQ    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Lys Asp Phe Gly Leu Pro Gln Glu Met Val Gl - #u Gly Asp Gln Leu Gln    #15    - Lys Asp Gln Ala Phe Pro Val Leu Tyr Glu Me - #t Leu Gln Gln Ser    #             30    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment 62-92    #ID NO:2       of SEQ    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Gln Gln Ser Phe Asn Leu Phe Tyr Thr Glu Hi - #s Ser Ser Ala Ala Trp    #15    - Asp Thr Thr Leu Leu Glu Gln Leu Cys Thr Gl - #y Leu Gln Gln Gln    #             30    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 33 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment 90-122    #ID NO:2       of SEQ    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Gln Gln Gln Leu Asp His Leu Asp Thr Cys Ar - #g Gly Gln Val Met Gly    #15    - Glu Glu Asp Ser Glu Leu Gly Asn Met Asp Pr - #o Ile Val Thr Val Lys    #             30    - Lys    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 32 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment:                   119-150 o - #f SEQ ID NO:2    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Thr Val Lys Lys Tyr Phe Gln Gly Ile Tyr As - #p Tyr Leu Gln Glu Lys    #15    - Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Ar - #g Val Glu Met Met Arg    #             30    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #amino acid sequence of fragment:                   139-172 o - #f SEQ ID NO:2    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - Cys Ala Trp Glu Ile Val Arg Val Glu Met Me - #t Arg Ala Leu Thr Val    #15    - Ser Thr Thr Leu Gln Lys Arg Leu Thr Lys Me - #t Gly Gly Asp Leu Asn    #             30    - Ser Pro    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 588 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau1 Human Interferon Tau coding    #with a leader sequence.    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..585    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - ATG GCC TTC GTG CTC TCT CTA CTC ATG GCC CT - #G GTG CTG GTC AGC TAC      48    Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - GGC CCA GGA GGA TCC CTG GGT TGT GAC CTG TC - #T CAG AAC CAC GTG CTG      96    Gly Pro Gly Gly Ser Leu Gly Cys Asp Leu Se - #r Gln Asn His Val Leu    #             30    - GTT GGC AGG AAG AAC CTC AGG CTC CTG GAC GA - #A ATG AGG AGA CTC TCC     144    Val Gly Arg Lys Asn Leu Arg Leu Leu Asp Gl - #u Met Arg Arg Leu Ser    #         45    - CCT CGC TTT TGT CTG CAG GAC AGA AAA GAC TT - #C GCT TTA CCC CAG GAA     192    Pro Arg Phe Cys Leu Gln Asp Arg Lys Asp Ph - #e Ala Leu Pro Gln Glu    #     60    - ATG GTG GAG GGC GGC CAG CTC CAG GAG GCC CA - #G GCC ATC TCT GTG CTC     240    Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gl - #n Ala Ile Ser Val Leu    # 80    - CAT GAG ATG CTC CAG CAG AGC TTC AAC CTC TT - #C CAC ACA GAG CAC TCC     288    His Glu Met Leu Gln Gln Ser Phe Asn Leu Ph - #e His Thr Glu His Ser    #                 95    - TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CA - #G CTC CGC ACT GGA CTC     336    Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gl - #n Leu Arg Thr Gly Leu    #           110    - CAT CAG CAG CTG GAC AAC CTG GAT GCC TGC CT - #G GGG CAG GTG ATG GGA     384    His Gln Gln Leu Asp Asn Leu Asp Ala Cys Le - #u Gly Gln Val Met Gly    #       125    - GAG GAA GAC TCT GCC CTG GGA AGG ACG GGC CC - #C ACC CTG GCT CTG AAG     432    Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Leu Lys    #   140    - AGG TAC TTC CAG GGC ATC CAT GTC TAC CTG AA - #A GAG AAG GGA TAC AGC     480    Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Ly - #s Glu Lys Gly Tyr Ser    145                 1 - #50                 1 - #55                 1 -    #60    - GAC TGC GCC TGG GAA ACC GTC AGA CTG GAA AT - #C ATG AGA TCC TTC TCT     528    Asp Cys Ala Trp Glu Thr Val Arg Leu Glu Il - #e Met Arg Ser Phe Ser    #               175    - TCA TTA ATC AGC TTG CAA GAA AGG TTA AGA AT - #G ATG GAT GGA GAC CTG     576    Ser Leu Ile Ser Leu Gln Glu Arg Leu Arg Me - #t Met Asp Gly Asp Leu    #           190    #      588    Ser Ser Pro            195    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 195 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:11 (HuIFNtau1).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - Gly Pro Gly Gly Ser Leu Gly Cys Asp Leu Se - #r Gln Asn His Val Leu    #             30    - Val Gly Arg Lys Asn Leu Arg Leu Leu Asp Gl - #u Met Arg Arg Leu Ser    #         45    - Pro Arg Phe Cys Leu Gln Asp Arg Lys Asp Ph - #e Ala Leu Pro Gln Glu    #     60    - Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gl - #n Ala Ile Ser Val Leu    # 80    - His Glu Met Leu Gln Gln Ser Phe Asn Leu Ph - #e His Thr Glu His Ser    #                 95    - Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gl - #n Leu Arg Thr Gly Leu    #           110    - His Gln Gln Leu Asp Asn Leu Asp Ala Cys Le - #u Gly Gln Val Met Gly    #       125    - Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Leu Lys    #   140    - Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Ly - #s Glu Lys Gly Tyr Ser    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Cys Ala Trp Glu Thr Val Arg Leu Glu Il - #e Met Arg Ser Phe Ser    #               175    - Ser Leu Ile Ser Leu Gln Glu Arg Leu Arg Me - #t Met Asp Gly Asp Leu    #           190    - Ser Ser Pro            195    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 25 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (synthetic)    -     (vi) ORIGINAL SOURCE:    #25-mer synthetic oligonucleotide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #               25 AAAA GACTT    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 25 bases              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (synthetic)    -     (vi) ORIGINAL SOURCE:    #25-mer synthetic oligonucleotide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #               25 TTCC CAGGC    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:4   1-37 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Arg Lys Asn Leu Arg    #                15    - Leu Leu Asp Glu Met Arg Arg Leu Ser Pro Ar - #g Phe Cys Leu Gln Asp    #            30    - Arg Lys Asp Phe Ala            35    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:4   34-64 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - Lys Asp Phe Ala Leu Pro Gln Glu Met Val Gl - #u Gly Gly Gln Leu Gln    #                15    - Glu Ala Gln Ala Ile Ser Val Leu His Glu Me - #t Leu Gln Gln Ser    #            30    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:4   62-92 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - Gln Gln Ser Phe Asn Leu Phe His Thr Glu Hi - #s Ser Ser Ala Ala Trp    #                15    - Asp Thr Thr Leu Leu Glu Gln Leu Arg Thr Gl - #y Leu His Gln Gln    #            30    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 33 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   90-122 of - # SEQ ID NO:4    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - His Gln Gln Leu Asp Asn Leu Asp Ala Cys Le - #u Gly Gln Val Met Gly    #                15    - Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Leu Lys    #            30    - Arg    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 32 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   119-150 o - #f SEQ ID NO:4    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - Ala Leu Lys Arg Tyr Phe Gln Gly Ile His Va - #l Tyr Leu Lys Glu Lys    #                15    - Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val Ar - #g Leu Glu Ile Met Arg    #            30    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   139-172 o - #f SEQ ID NO:4    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    - Cys Ala Trp Glu Thr Val Arg Leu Glu Ile Me - #t Arg Ser Phe Ser Ser    #                15    - Leu Ile Ser Leu Gln Glu Arg Leu Arg Met Me - #t Asp Gly Asp Leu Ser    #            30    - Ser Pro    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 299 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau6(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 2..298    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    #GAG GCC CAG GCC ATC         46 CAG CTC CAG      Gln Glu Met Val Glu Gly Gly Gln Leu G - #ln Glu Ala Gln Ala Ile    # 15    - TCT GTG CTC CAC AAG ATG CTC CAG CAG AGC TT - #C AAC CTC TTC CAC ACA      94    Ser Val Leu His Lys Met Leu Gln Gln Ser Ph - #e Asn Leu Phe His Thr    #                 30    - GAG CGC TCC TCT GCT GCC TGG GAC ACC ACC CT - #C CTG GAG CAG CTC CGC     142    Glu Arg Ser Ser Ala Ala Trp Asp Thr Thr Le - #u Leu Glu Gln Leu Arg    #             45    - ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG GA - #C GCC TGC CTG GGG CAG     190    Thr Gly Leu His Gln Gln Leu Asp Asp Leu As - #p Ala Cys Leu Gly Gln    #         60    - GTG ACG GGA GAG GAA GAC TCT GCC CTG GGA AG - #G ACG GGC CCC ACC CTG     238    Val Thr Gly Glu Glu Asp Ser Ala Leu Gly Ar - #g Thr Gly Pro Thr Leu    #     75    - GCC GTG AAG AGC TAC TTC CAG GGC ATC CAT AT - #C TAC CTG CAA GAG AAG     286    Ala Val Lys Ser Tyr Phe Gln Gly Ile His Il - #e Tyr Leu Gln Glu Lys    # 95    #     299    Gly Tyr Ser Asp    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:21 (HuIFNtau6).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    - Gln Glu Met Val Glu Gly Gly Gln Leu Gln Gl - #u Ala Gln Ala Ile Ser    #                 15    - Val Leu His Lys Met Leu Gln Gln Ser Phe As - #n Leu Phe His Thr Glu    #             30    - Arg Ser Ser Ala Ala Trp Asp Thr Thr Leu Le - #u Glu Gln Leu Arg Thr    #         45    - Gly Leu His Gln Gln Leu Asp Asp Leu Asp Al - #a Cys Leu Gly Gln Val    #     60    - Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Th - #r Gly Pro Thr Leu Ala    # 80    - Val Lys Ser Tyr Phe Gln Gly Ile His Ile Ty - #r Leu Gln Glu Lys Gly    #                 95    - Tyr Ser Asp    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 288 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau7(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 2..286    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    #GAG GCC CAG GCC ATT         46 CAG TTC CAG      Gln Glu Met Val Glu Val Ser Gln Phe G - #ln Glu Ala Gln Ala Ile    # 15    - TCT GTG CTC CAT GAG ATG CTC CAG CAG AGC TT - #C AAC CTC TTC CAC AAA      94    Ser Val Leu His Glu Met Leu Gln Gln Ser Ph - #e Asn Leu Phe His Lys    #                 30    - GAG CGC TCC TCT GCT GCC TGG GAC ACT ACC CT - #C CTG GAG CAG CTC CTC     142    Glu Arg Ser Ser Ala Ala Trp Asp Thr Thr Le - #u Leu Glu Gln Leu Leu    #             45    - ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG GA - #T GCC TGT CTG GGG CAG     190    Thr Gly Leu His Gln Gln Leu Asp Asp Leu As - #p Ala Cys Leu Gly Gln    #         60    - TTG ACT GGA GAG GAA GAC TCT GCC CTG GGA AG - #G ACG GGC CCC ACC CTG     238    Leu Thr Gly Glu Glu Asp Ser Ala Leu Gly Ar - #g Thr Gly Pro Thr Leu    #     75    - GCC GTG AAG AGC TAC TTC CAG GGC ATC CAT GT - #C TAC CTG CAA GAG AAG     286    Ala Val Lys Ser Tyr Phe Gln Gly Ile His Va - #l Tyr Leu Gln Glu Lys    # 95    #             288    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 95 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:23 (HuIFNtau7).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - Gln Glu Met Val Glu Val Ser Gln Phe Gln Gl - #u Ala Gln Ala Ile Ser    #                 15    - Val Leu His Glu Met Leu Gln Gln Ser Phe As - #n Leu Phe His Lys Glu    #             30    - Arg Ser Ser Ala Ala Trp Asp Thr Thr Leu Le - #u Glu Gln Leu Leu Thr    #         45    - Gly Leu His Gln Gln Leu Asp Asp Leu Asp Al - #a Cys Leu Gly Gln Leu    #     60    - Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Th - #r Gly Pro Thr Leu Ala    # 80    - Val Lys Ser Tyr Phe Gln Gly Ile His Val Ty - #r Leu Gln Glu Lys    #                 95    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 307 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau4(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 2..307    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #GAG GCC CAG GCC ATC         46 CAG CTC CAG      Gln Glu Met Val Glu Gly Gly Gln Leu G - #ln Glu Ala Gln Ala Ile    # 15    - TCT GTG CTC CAC GAG ATG CTC CAG CAG AGC TT - #C AAC CTC TTC CAC ACA      94    Ser Val Leu His Glu Met Leu Gln Gln Ser Ph - #e Asn Leu Phe His Thr    #                 30    - GAG CAC TCC TCT GCT GCC TGG GAC ACC ACC CT - #C CTG GAG CAG CTC CGC     142    Glu His Ser Ser Ala Ala Trp Asp Thr Thr Le - #u Leu Glu Gln Leu Arg    #             45    - ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG GA - #T GCC TGC CTG GGG CAG     190    Thr Gly Leu His Gln Gln Leu Asp Asp Leu As - #p Ala Cys Leu Gly Gln    #         60    - GTG ACG GGA GAG GAA GAC TCT GCC CTG GGA AG - #G ACG GGC CCC ACC CTG     238    Val Thr Gly Glu Glu Asp Ser Ala Leu Gly Ar - #g Thr Gly Pro Thr Leu    #     75    - GCC ATG AAG ACG TAT TTC CAG GGC ATC CAT GT - #C TAC CTG AAA GAG AAG     286    Ala Met Lys Thr Tyr Phe Gln Gly Ile His Va - #l Tyr Leu Lys Glu Lys    # 95    #                 307 GCC TGG    Gly Tyr Ser Asp Cys Ala Trp                    100    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 102 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:25 (HuIFNtau4).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    - Gln Glu Met Val Glu Gly Gly Gln Leu Gln Gl - #u Ala Gln Ala Ile Ser    #                 15    - Val Leu His Glu Met Leu Gln Gln Ser Phe As - #n Leu Phe His Thr Glu    #             30    - His Ser Ser Ala Ala Trp Asp Thr Thr Leu Le - #u Glu Gln Leu Arg Thr    #         45    - Gly Leu His Gln Gln Leu Asp Asp Leu Asp Al - #a Cys Leu Gly Gln Val    #     60    - Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Th - #r Gly Pro Thr Leu Ala    # 80    - Met Lys Thr Tyr Phe Gln Gly Ile His Val Ty - #r Leu Lys Glu Lys Gly    #                 95    - Tyr Ser Asp Cys Ala Trp                100    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 294 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau5(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 2..292    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #GAG GCC CAG GCC ATC         46 CAG CTC CAG      Gln Glu Met Val Glu Gly Gly Gln Leu G - #ln Glu Ala Gln Ala Ile    # 15    - TCT GTG CTC CAC GAG ATG CTC CAG CAG AGC TT - #C AAC CTC TTC CAC ACA      94    Ser Val Leu His Glu Met Leu Gln Gln Ser Ph - #e Asn Leu Phe His Thr    #                 30    - GAG CAC TCC TCT GCT GCC TGG GAC ACC ACC CT - #C CTG GAG CAG CTC CGC     142    Glu His Ser Ser Ala Ala Trp Asp Thr Thr Le - #u Leu Glu Gln Leu Arg    #             45    - ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG GA - #T GCC TGC CTG GGG CAG     190    Thr Gly Leu His Gln Gln Leu Asp Asp Leu As - #p Ala Cys Leu Gly Gln    #         60    - GTG ACG GGA GAG GAA GAC TCT GCC CTG GGA AG - #G ACG GGC CCC ACC CTG     238    Val Thr Gly Glu Glu Asp Ser Ala Leu Gly Ar - #g Thr Gly Pro Thr Leu    #     75    - GCC ATG AAG ACG TAT TTC CAG GGC ATC CAT GT - #C TAC CTG AAA GAG AAG     286    Ala Met Lys Thr Tyr Phe Gln Gly Ile His Va - #l Tyr Leu Lys Glu Lys    # 95    #         294    Gly Tyr    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 97 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:27 (HuIFNtau5).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    - Gln Glu Met Val Glu Gly Gly Gln Leu Gln Gl - #u Ala Gln Ala Ile Ser    #                 15    - Val Leu His Glu Met Leu Gln Gln Ser Phe As - #n Leu Phe His Thr Glu    #             30    - His Ser Ser Ala Ala Trp Asp Thr Thr Leu Le - #u Glu Gln Leu Arg Thr    #         45    - Gly Leu His Gln Gln Leu Asp Asp Leu Asp Al - #a Cys Leu Gly Gln Val    #     60    - Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Th - #r Gly Pro Thr Leu Ala    # 80    - Met Lys Thr Tyr Phe Gln Gly Ile His Val Ty - #r Leu Lys Glu Lys Gly    #                 95    - Tyr    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 516 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau2(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..516    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    - GAC CTG TCT CAG AAC CAC GTG CTG GTT GGC AG - #G AAG AAC CTC AGG CTC      48    Asp Leu Ser Gln Asn His Val Leu Val Gly Ar - #g Lys Asn Leu Arg Leu    #                 15    - CTG GAC CAA ATG AGG AGA CTC TCC CCT CGC TT - #T TGT CTG CAG GAC AGA      96    Leu Asp Gln Met Arg Arg Leu Ser Pro Arg Ph - #e Cys Leu Gln Asp Arg    #             30    - AAA GAC TTC GCT TTA CCC TAG GAA ATG GTG GA - #G GGC GGC CAG CTC CAG     144    #Val Glu Gly Gly Gln Leu GlnGlu Met    #         45    - GAG GCC CAG GCC ATC TCT GTG CTC CAT GAG AT - #G CTC CAG CAG AGC TTC     192    Glu Ala Gln Ala Ile Ser Val Leu His Glu Me - #t Leu Gln Gln Ser Phe    #     60    - AAC CTC TTC CAC ACA GAG CAC TCC TCT GCT GC - #C TGG GAC ACC ACC CTC     240    Asn Leu Phe His Thr Glu His Ser Ser Ala Al - #a Trp Asp Thr Thr Leu    # 80    - CTG GAG CAG CTC CGC ACT GGA CTC CAT CAG CA - #G CTG GAC AAC CTG GAT     288    Leu Glu Gln Leu Arg Thr Gly Leu His Gln Gl - #n Leu Asp Asn Leu Asp    #                 95    - GCC TGC CTG GGG CAG GTG ATG GGA GAG GAA GA - #C TCT GCC CTG GGA AGG     336    Ala Cys Leu Gly Gln Val Met Gly Glu Glu As - #p Ser Ala Leu Gly Arg    #           110    - ACG GGC CCC ACC CTG GCT CTG AAG AGG TAC TT - #C CAG GGC ATC CAT GTC     384    Thr Gly Pro Thr Leu Ala Leu Lys Arg Tyr Ph - #e Gln Gly Ile His Val    #       125    - TAC CTG AAA GAG AAG GGA TAC AGC GAC TGC GC - #C TGG GAA ACC GTC AGA     432    Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Al - #a Trp Glu Thr Val Arg    #   140    - GTG GAA ATC ATG AGA TCC TTC TCT TCA TTA AT - #C AGC TTG CAA GAA AGG     480    Val Glu Ile Met Arg Ser Phe Ser Ser Leu Il - #e Ser Leu Gln Glu Arg    145                 1 - #50                 1 - #55                 1 -    #60    #      516ATG ATG GAT GGA GAC CTG AGC TCA CC - #T TGA    Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pr - #o    #               170    -         (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 115-117    #/note= "to allow expression of the    #protein this site can be    #to encode an amino acid, e.g.,    #               Gln"    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 171 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:29 (HuIFNtau2).    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    - Asp Leu Ser Gln Asn His Val Leu Val Gly Ar - #g Lys Asn Leu Arg Leu    #                 15    - Leu Asp Gln Met Arg Arg Leu Ser Pro Arg Ph - #e Cys Leu Gln Asp Arg    #             30    - Lys Asp Phe Ala Leu Pro Xaa Glu Met Val Gl - #u Gly Gly Gln Leu Gln    #         45    - Glu Ala Gln Ala Ile Ser Val Leu His Glu Me - #t Leu Gln Gln Ser Phe    #     60    - Asn Leu Phe His Thr Glu His Ser Ser Ala Al - #a Trp Asp Thr Thr Leu    # 80    - Leu Glu Gln Leu Arg Thr Gly Leu His Gln Gl - #n Leu Asp Asn Leu Asp    #                 95    - Ala Cys Leu Gly Gln Val Met Gly Glu Glu As - #p Ser Ala Leu Gly Arg    #           110    - Thr Gly Pro Thr Leu Ala Leu Lys Arg Tyr Ph - #e Gln Gly Ile His Val    #       125    - Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Al - #a Trp Glu Thr Val Arg    #   140    - Val Glu Ile Met Arg Ser Phe Ser Ser Leu Il - #e Ser Leu Gln Glu Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pr - #o    #               170    -         (ix) FEATURE:              (A) NAME/KEY: Modified-sit - #e              (B) LOCATION: 39    #/note= "where Xaa a selected amino    #               acid for - # example, Gln"    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 588 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau3(C) INDIVIDUAL ISOLATE:    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..588    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    - ATG GCC TTC GTG CTC TCT CTA CTC ATG GCC CT - #G GTG CTG GTC AGC TAC      48    Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - GGC CCG GGA GGA TCC CTG CGG TGT GAC CTG TC - #T CAG AAC CAC GTG CTG      96    Gly Pro Gly Gly Ser Leu Arg Cys Asp Leu Se - #r Gln Asn His Val Leu    #             30    - GTT GGC AGC CAG AAC CTC AGG CTC CTG GGC CA - #A ATG AGG AGA CTC TCC     144    Val Gly Ser Gln Asn Leu Arg Leu Leu Gly Gl - #n Met Arg Arg Leu Ser    #         45    - CTT CGC TTC TGT CTG CAG GAC AGA AAA GAC TT - #C GCT TTC CCC CAG GAG     192    Leu Arg Phe Cys Leu Gln Asp Arg Lys Asp Ph - #e Ala Phe Pro Gln Glu    #     60    - ATG GTG GAG GGT GGC CAG CTC CAG GAG GCC CA - #G GCC ATC TCT GTG CTC     240    Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gl - #n Ala Ile Ser Val Leu    # 80    - CAC GAG ATG CTC CAG CAG AGC TTC AAC CTC TT - #C CAC ACA GAG CAC TCC     288    His Glu Met Leu Gln Gln Ser Phe Asn Leu Ph - #e His Thr Glu His Ser    #                 95    - TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CA - #G CTC CGC ACT GGA CTC     336    Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gl - #n Leu Arg Thr Gly Leu    #           110    - CAT CAG CAG CTG GAT GAC CTG GAT GCC TGC CT - #G GGG CAG GTG ACG GGA     384    His Gln Gln Leu Asp Asp Leu Asp Ala Cys Le - #u Gly Gln Val Thr Gly    #       125    - GAG GAA GAC TCT GCC CTG GGA AGA ACG GGC CC - #C ACC CTG GCC ATG AAG     432    Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Met Lys    #   140    - AGG TAT TTC CAG GGC ATC CAT GTC TAC CTG AA - #A GAG AAG GGA TAT AGT     480    Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Ly - #s Glu Lys Gly Tyr Ser    145                 1 - #50                 1 - #55                 1 -    #60    - GAC TGC GCC TGG GAA ATT GTC AGA CTG GAA AT - #C ATG AGA TCC TTG TCT     528    Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Il - #e Met Arg Ser Leu Ser    #               175    - TCA TCA ACC AGC TTG CAC AAA AGG TTA AGA AT - #G ATG GAT GGA GAC CTG     576    Ser Ser Thr Ser Leu His Lys Arg Leu Arg Me - #t Met Asp Gly Asp Leu    #           190    #      588    Ser Ser Pro            195    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 195 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #predicted amino acid coding sequence    #ID NO:31 (HuIFNtau3)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    - Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - Gly Pro Gly Gly Ser Leu Arg Cys Asp Leu Se - #r Gln Asn His Val Leu    #             30    - Val Gly Ser Gln Asn Leu Arg Leu Leu Gly Gl - #n Met Arg Arg Leu Ser    #         45    - Leu Arg Phe Cys Leu Gln Asp Arg Lys Asp Ph - #e Ala Phe Pro Gln Glu    #     60    - Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gl - #n Ala Ile Ser Val Leu    # 80    - His Glu Met Leu Gln Gln Ser Phe Asn Leu Ph - #e His Thr Glu His Ser    #                 95    - Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gl - #n Leu Arg Thr Gly Leu    #           110    - His Gln Gln Leu Asp Asp Leu Asp Ala Cys Le - #u Gly Gln Val Thr Gly    #       125    - Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Met Lys    #   140    - Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Ly - #s Glu Lys Gly Tyr Ser    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Il - #e Met Arg Ser Leu Ser    #               175    - Ser Ser Thr Ser Leu His Lys Arg Leu Arg Me - #t Met Asp Gly Asp Leu    #           190    - Ser Ser Pro            195    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 518 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau3, mature no leader sequence    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..518    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    - TGT GAC CTG TCT CAG AAC CAC GTG CTG GTT GG - #C AGC CAG AAC CTC AGG      48    Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Ser Gln Asn Leu Arg    #                 15    - CTC CTG GGC CAA ATG AGG AGA CTC TCC CTT CG - #C TTC TGT CTG CAG GAC      96    Leu Leu Gly Gln Met Arg Arg Leu Ser Leu Ar - #g Phe Cys Leu Gln Asp    #             30    - AGA AAA GAC TTC GCT TTC CCC CAG GAG ATG GT - #G GAG GGT GGC CAG CTC     144    Arg Lys Asp Phe Ala Phe Pro Gln Glu Met Va - #l Glu Gly Gly Gln Leu    #         45    - CAG GAG GCC CAG GCC ATC TCT GTG CTC CAC GA - #G ATG CTC CAG CAG AGC     192    Gln Glu Ala Gln Ala Ile Ser Val Leu His Gl - #u Met Leu Gln Gln Ser    #     60    - TTC AAC CTC TTC CAC ACA GAG CAC TCC TCT GC - #T GCC TGG GAC ACC ACC     240    Phe Asn Leu Phe His Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - CTC CTG GAG CAG CTC CGC ACT GGA CTC CAT CA - #G CAG CTG GAT GAC CTG     288    Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gl - #n Gln Leu Asp Asp Leu    #                 95    - GAT GCC TGC CTG GGG CAG GTG ACG GGA GAG GA - #A GAC TCT GCC CTG GGA     336    Asp Ala Cys Leu Gly Gln Val Thr Gly Glu Gl - #u Asp Ser Ala Leu Gly    #           110    - AGA ACG GGC CCC ACC CTG GCC ATG AAG AGG TA - #T TTC CAG GGC ATC CAT     384    Arg Thr Gly Pro Thr Leu Ala Met Lys Arg Ty - #r Phe Gln Gly Ile His    #       125    - GTC TAC CTG AAA GAG AAG GGA TAT AGT GAC TG - #C GCC TGG GAA ATT GTC     432    Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Ile Val    #   140    - AGA CTG GAA ATC ATG AGA TCC TTG TCT TCA TC - #A ACC AGC TTG CAC AAA     480    Arg Leu Glu Ile Met Arg Ser Leu Ser Ser Se - #r Thr Ser Leu His Lys    145                 1 - #50                 1 - #55                 1 -    #60    #    518A AGA ATG ATG GAT GGA GAC CTG AGC TC - #A CCT TG    Arg Leu Arg Met Met Asp Gly Asp Leu Ser Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 172 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    - Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Ser Gln Asn Leu Arg    #                 15    - Leu Leu Gly Gln Met Arg Arg Leu Ser Leu Ar - #g Phe Cys Leu Gln Asp    #             30    - Arg Lys Asp Phe Ala Phe Pro Gln Glu Met Va - #l Glu Gly Gly Gln Leu    #         45    - Gln Glu Ala Gln Ala Ile Ser Val Leu His Gl - #u Met Leu Gln Gln Ser    #     60    - Phe Asn Leu Phe His Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gl - #n Gln Leu Asp Asp Leu    #                 95    - Asp Ala Cys Leu Gly Gln Val Thr Gly Glu Gl - #u Asp Ser Ala Leu Gly    #           110    - Arg Thr Gly Pro Thr Leu Ala Met Lys Arg Ty - #r Phe Gln Gly Ile His    #       125    - Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Ile Val    #   140    - Arg Leu Glu Ile Met Arg Ser Leu Ser Ser Se - #r Thr Ser Leu His Lys    145                 1 - #50                 1 - #55                 1 -    #60    - Arg Leu Arg Met Met Asp Gly Asp Leu Ser Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:33  1-37 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    - Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Ser Gln Asn Leu Arg    #                 15    - Leu Leu Gly Gln Met Arg Arg Leu Ser Leu Ar - #g Phe Cys Leu Gln Asp    #             30    - Arg Lys Asp Phe Ala             35    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:33  34-64 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    - Lys Asp Phe Ala Phe Pro Gln Glu Met Val Gl - #u Gly Gly Gln Leu Gln    #                 15    - Glu Ala Gln Ala Ile Ser Val Leu His Glu Me - #t Leu Gln Gln Ser    #             30    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 31 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:33  62-92 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    - Gln Gln Ser Phe Asn Leu Phe His Thr Glu Hi - #s Ser Ser Ala Ala Trp    #                 15    - Asp Thr Thr Leu Leu Glu Gln Leu Arg Thr Gl - #y Leu His Gln Gln    #             30    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 33 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   90-122 of - # SEQ ID NO:33    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    - His Gln Gln Leu Asp Asp Leu Asp Ala Cys Le - #u Gly Gln Val Thr Gly    #                 15    - Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pr - #o Thr Leu Ala Met Lys    #             30    - Arg    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 32 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   119-150 o - #f SEQ ID NO:33    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    - Ala Met Lys Arg Tyr Phe Gln Gly Ile His Va - #l Tyr Leu Lys Glu Lys    #                 15    - Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val Ar - #g Leu Glu Ile Met Arg    #             30    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:                   139-172 o - #f SEQ ID NO:33    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    - Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Me - #t Arg Ser Leu Ser Ser    #                 15    - Ser Thr Ser Leu His Lys Arg Leu Arg Met Me - #t Asp Gly Asp Leu Ser    #             30    - Ser Pro    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 23 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:32  1-23 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    - Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - Gly Pro Gly Gly Ser Leu Arg                 20    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 23 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:    #Amino acid sequence of fragment:    #SEQ ID NO:11  1-23 of    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    - Met Ala Phe Val Leu Ser Leu Leu Met Ala Le - #u Val Leu Val Ser Tyr    #                 15    - Gly Pro Gly Gly Ser Leu Gly                 20    - (2) INFORMATION FOR SEQ ID NO:43:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 519 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #HuIFNtau1 genomic-derivedSOLATE:                   DNA codin - #g sequence, without leader seq.    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..519    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    - TGT GAC CTG TCT CAG AAC CAC GTG CTG GTT GG - #C AGG AAG AAC CTC AGG      48    Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Arg Lys Asn Leu Arg    #                 15    - CTC CTG GAC GAA ATG AGG AGA CTC TCC CCT CG - #C TTT TGT CTG CAG GAC      96    Leu Leu Asp Glu Met Arg Arg Leu Ser Pro Ar - #g Phe Cys Leu Gln Asp    #             30    - AGA AAA GAC TTC GCT TTA CCC CAG GAA ATG GT - #G GAG GGC GGC CAG CTC     144    Arg Lys Asp Phe Ala Leu Pro Gln Glu Met Va - #l Glu Gly Gly Gln Leu    #         45    - CAG GAG GCC CAG GCC ATC TCT GTG CTC CAT GA - #G ATG CTC CAG CAG AGC     192    Gln Glu Ala Gln Ala Ile Ser Val Leu His Gl - #u Met Leu Gln Gln Ser    #     60    - TTC AAC CTC TTC CAC ACA GAG CAC TCC TCT GC - #T GCC TGG GAC ACC ACC     240    Phe Asn Leu Phe His Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - CTC CTG GAG CAG CTC CGC ACT GGA CTC CAT CA - #G CAG CTG GAC AAC CTG     288    Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gl - #n Gln Leu Asp Asn Leu    #                 95    - GAT GCC TGC CTG GGG CAG GTG ATG GGA GAG GA - #A GAC TCT GCC CTG GGA     336    Asp Ala Cys Leu Gly Gln Val Met Gly Glu Gl - #u Asp Ser Ala Leu Gly    #           110    - AGG ACG GGC CCC ACC CTG GCT CTG AAG AGG TA - #C TTC CAG GGC ATC CAT     384    Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Ty - #r Phe Gln Gly Ile His    #       125    - GTC TAC CTG AAA GAG AAG GGA TAC AGC GAC TG - #C GCC TGG GAA ACC GTC     432    Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Thr Val    #   140    - AGA CTG GAA ATC ATG AGA TCC TTC TCT TCA TT - #A ATC AGC TTG CAA GAA     480    Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Le - #u Ile Ser Leu Gln Glu    145                 1 - #50                 1 - #55                 1 -    #60    #    519A AGA ATG ATG GAT GGA GAC CTG AGC TC - #A CCT TGA    Arg Leu Arg Met Met Asp Gly Asp Leu Ser Se - #r Pro    #               170    - (2) INFORMATION FOR SEQ ID NO:44:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 172 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    - Cys Asp Leu Ser Gln Asn His Val Leu Val Gl - #y Arg Lys Asn Leu Arg    #                 15    - Leu Leu Asp Glu Met Arg Arg Leu Ser Pro Ar - #g Phe Cys Leu Gln Asp    #             30    - Arg Lys Asp Phe Ala Leu Pro Gln Glu Met Va - #l Glu Gly Gly Gln Leu    #         45    - Gln Glu Ala Gln Ala Ile Ser Val Leu His Gl - #u Met Leu Gln Gln Ser    #     60    - Phe Asn Leu Phe His Thr Glu His Ser Ser Al - #a Ala Trp Asp Thr Thr    # 80    - Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gl - #n Gln Leu Asp Asn Leu    #                 95    - Asp Ala Cys Leu Gly Gln Val Met Gly Glu Gl - #u Asp Ser Ala Leu Gly    #           110    - Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Ty - #r Phe Gln Gly Ile His    #       125    - Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cy - #s Ala Trp Glu Thr Val    #   140    - Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Le - #u Ile Ser Leu Gln Glu    145                 1 - #50                 1 - #55                 1 -    #60    - Arg Leu Arg Met Met Asp Gly Asp Leu Ser Se - #r Pro    #               170    __________________________________________________________________________

We claim:
 1. A method of inhibiting growth of tumor cells in a subject,comprisingadministering ovine or bovine interferon-tau to the subject inan amount effective to inhibit growth of the tumor cells and whichinduces a change in white blood cell count in the subject which is lessthan the change in white blood cell count which would be induced in thesubject by an amount of interferon-alpha having the same tumor growthinhibition effect.
 2. The method of claim 1, where said interferon-tauhas the amino acid sequence presented as SEQ ID NO:2.
 3. The method ofclaim 1, wherein said tumor cells are selected from the group consistingof human carcinoma cells, human leukemia cells, human T-lymphoma cells,and human melanoma cells.
 4. The method of claim 3, wherein said tumorcells are selected from the group consisting of human lung large cellcarcinoma, human colon adenocarcinoma, human malignant melanoma, humanrenal adenocarcinoma, human promyelocytic leukemia, human T celllymphoma, human cutaneous T cell lymphoma, and human breast adenocinoma.5. The method of claim 3, wherein said tumor cells are steroid-sensitivetumor cells.
 6. The method of claim 3, wherein said tumor cells aremammary tumor cells.